http://guidelines.beefimprovement.org/api.php?action=feedcontributions&user=AlisonVE&feedformat=atomBIF Guidelines Wiki - User contributions [en]2024-03-29T02:13:51ZUser contributionsMediaWiki 1.35.2http://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2517Data From Gene Edited Animals2022-06-03T16:55:50Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
Additionally, in March 2022 the FDA announced it planned to exercise enforcement discretion for the marketing of products, including food, from two of Acceligen Inc.’s “PRLR-SLICK” genome-edited beef cattle and their offspring after determining that the IGA (i.e. prolactin receptor mutation) did not raise any safety concerns (i.e. low-risk determination). This was first low-risk determination for enforcement discretion for an IGA in an animal for food use. What this means in practice is that, while developers are generally required to have an approved new animal drug application for IGAs in animals prior to marketing, on a case-by-case basis for those edits that are low risk, the FDA may not expect developers to seek approval of these IGAs.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change) and phenotype<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing reagents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and the results (e.g. heterozygous Pp) should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2516Data From Gene Edited Animals2022-06-03T16:52:48Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
It should be noted that in March 2022 the FDA announced it planned to exercise enforcement discretion for the marketing of products, including food, from two of Acceligen Inc.’s “PRLR-SLICK” genome-edited beef cattle and their offspring after determining that the intentional genomic alteration (IGA) which in this specific case was prolactin receptor mutations did not raise any safety concerns (i.e. low-risk determination). This was first low-risk determination for enforcement discretion for an IGA in an animal for food use. What this means in practice is that, while developers are generally required to have an approved new animal drug application for IGAs in animals prior to marketing, on a case-by-case basis for those edits that are low risk, the FDA may not expect developers to seek approval of these IGAs.<br />
<br />
The data that was provided to the FDA to obtain this low risk determination is detailed in the risk assessment summary and included “genomic data and other information to FDA to demonstrate that the IGA contained in PRLR-SLICK cattle is the equivalent to naturally occurring mutations that occur in<br />
conventionally raised cattle with a history of safe use as a source of human food”. <br />
<br />
According to this document, “Acceligen provided raw whole genome sequencing (WGS) data and their bioinformatics analysis for four edited calves and their unedited parents. This genotypic data was used to evaluate the actual sequences of the IGA in the animals’ genomes and screen for the presence of any unintended alterations that might pose an animal or food safety concern and risk to the environment. FDA independently analyzed the WGS data and confirmed Acceligen’s conclusion that of the four calves born, three contained the IGA in exon 9 of the PRLR gene and one was determined to be unedited (“no-take”). [One of the edited animals subsequently died of bovine congestive heart failure; BCHF]. Acceligen’s and FDA’s analyses showed that the alleles containing the IGA contain premature stop codons that would result in truncated PRLR protein analogous to the naturally occurring slick mutations. Due to the specific process used to generate the IGA, the cattle containing the IGA were mosaic, meaning that in different cells/tissues of the cattle, different alleles may be present (these cattle could have several, including multiple distinct but equivalent IGA alleles as well as wild-type or otherwise non-IGA alleles). Acceligen included a disclaimer in the product label to describe that PRLR-SLICK cattle may have 2 or more genetically different sets of cells and, as a result, first-generation progeny may not all inherit the slick phenotype.<br />
<br />
From the WGS data, there was evidence of unintended alterations in the cattle containing the IGA. FDA’s analysis confirmed the unintended alterations reported by Acceligen and found evidence for a few additional unintended alterations, including a duplication located in a repetitive intergenic region and indels (short insertions and deletions) in intergenic regions. Except for the duplication, all unintended alterations are indels ranging from 1 to 11 DNA base pairs in length located in intronic or intergenic regions. The discrepancy between FDA’s and Acceligen’s analysis results are attributable to differences in bioinformatics analysis. None of the identified unintended alterations are expected to result in changes to protein expression based on their locations and available genome annotation. Based on the molecular characterization and animal health data, FDA determined these unintended alterations do not pose any safety concerns for the cattle or for humans consuming food from the cattle. <br />
<br />
Based on the data and other information submitted by Acceligen, FDA determined that the IGA contained in PRLR-SLICK cattle and the cattle’s associated products, including offspring, semen, embryos, and food products derived from them, pose low risk to people, animals, the food supply, and the environment. Therefore, FDA does not intend to object to Acceligen marketing the IGA in PRLR-SLICK cattle or marketing the cattle’s associated products. FDA also does not intend to object to Acceligen introducing cattle containing the IGA into the food supply. The agency’s decision is limited to the marketed products (for example, live PRLRSLICK cattle, semen, embryos, and meat) derived from the existing two cattle containing the IGA for which FDA has reviewed data and their progeny. Additionally, FDA intends to treat facilities or farms that are engaged in standard agricultural practices for PRLR- SLICK cattle, including assisted reproduction techniques (such as embryo transfer) or raising them for food<br />
production, the same as facilities or farms that are engaged in these practices for cattle without IGAs.”<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change) and phenotype<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing reagents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and the results (e.g. heterozygous Pp) should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2474Data From Gene Edited Animals2021-10-09T16:23:08Z<p>AlisonVE: </p>
<hr />
<div>[[Category:Data Collection]]<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change) and phenotype<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing reagents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and the results (e.g. heterozygous Pp) should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2473Data From Gene Edited Animals2021-09-30T19:23:48Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change) and phenotype<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing reagents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and the results (e.g. heterozygous Pp) should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2472Data From Gene Edited Animals2021-09-28T17:03:24Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing reagents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and the results (e.g. heterozygous Pp) should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2471Data From Gene Edited Animals2021-09-28T15:44:43Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and the results (e.g. heterozygous Pp) should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2470Data From Gene Edited Animals2021-09-28T15:43:11Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) <br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and test results should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2469Data From Gene Edited Animals2021-09-28T15:42:32Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2) and test results should be printed on registration certificates<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and test results should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2468Data From Gene Edited Animals2021-09-28T15:41:00Z<p>AlisonVE: /* Recommendations */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
* ''The offspring of a genome edited animal should be genotyped for inheritance of one or two copies of the intended alteration in the same way as animals are genotyped for other genetic conditions and test results should be printed on registration certificates.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2467Data From Gene Edited Animals2021-09-28T15:27:43Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal at the intentional alteration (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2466Data From Gene Edited Animals2021-09-28T15:24:28Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2465Data From Gene Edited Animals2021-09-28T15:23:35Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing reagents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2464Data From Gene Edited Animals2021-09-28T15:23:16Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients as those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2463Data From Gene Edited Animals2021-09-23T23:50:46Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. https://doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2462Data From Gene Edited Animals2021-09-23T23:50:22Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. https://doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 https://doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. https://doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. https://doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2461Data From Gene Edited Animals2021-09-23T23:49:19Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref><ref>Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R, Bendiek J, Sprink T., 2019. Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236. https://doi.org/10.3389/fpls.2019.00236</ref><br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2460Data From Gene Edited Animals2021-09-18T23:28:49Z<p>AlisonVE: /* Data Recording */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the intended alteration(s) were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the intended alteration(s) that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the intended alteration(s) and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2459Data From Gene Edited Animals2021-09-18T18:42:49Z<p>AlisonVE: </p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT ARTICLE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2458Data From Gene Edited Animals2021-09-18T18:34:39Z<p>AlisonVE: </p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that a study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2457Data From Gene Edited Animals2021-09-18T17:57:23Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''[https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-187-regulation-intentionally-altered-genomic-dna-animals are regulated as new animal drugs by the FDA].''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline ([http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCXG%2B68-2008%252FCXG_068e.pdf CAC/CL 68-2008]) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2456Data From Gene Edited Animals2021-09-18T17:54:49Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''are regulated as new animal drugs by the FDA.''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline (CAC/CL 68-2008) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2455Data From Gene Edited Animals2021-09-18T17:53:50Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[[File:Picture1.jpg]]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype <ref name="BandE"></ref>.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''are regulated as new animal drugs by the FDA.''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline (CAC/CL 68-2008) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2454Data From Gene Edited Animals2021-09-18T17:52:38Z<p>AlisonVE: /* Genome or Gene Editing */</p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
<br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
<br />
[File:Picture1.jpg]<br />
<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype <ref name="BandE"></ref>.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''are regulated as new animal drugs by the FDA.''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline (CAC/CL 68-2008) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=File:Picture1.jpg&diff=2453File:Picture1.jpg2021-09-18T17:50:30Z<p>AlisonVE: </p>
<hr />
<div>This shows two types of repair pathways for double stranded genome breaks</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2452Data From Gene Edited Animals2021-09-18T15:35:24Z<p>AlisonVE: </p>
<hr />
<div>[[Category:Data Collection]]<br />
<center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
[[File:GE_Figure1.png]]<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype <ref name="BandE"></ref>.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''are regulated as new animal drugs by the FDA.''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline (CAC/CL 68-2008) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Data_From_Gene_Edited_Animals&diff=2451Data From Gene Edited Animals2021-09-18T15:22:01Z<p>AlisonVE: Created page with "<center> '''THIS IS A DRAFT PAGE PENDING APPROVAL''' </center> =Genome or Gene Editing= Genome editing offers an approach to introduce targeted edits into the genome. It can b..."</p>
<hr />
<div><center><br />
'''THIS IS A DRAFT PAGE PENDING APPROVAL'''<br />
</center><br />
=Genome or Gene Editing=<br />
Genome editing offers an approach to introduce targeted edits into the genome. It can be used to introduce targeted knock-outs of specific genes, or perform intraspecies allele substitutions using a donor template with endogenous DNA sequences. Both of these cases do not introduce novel DNA and resemble genetic variations that can be found in the cattle genome. It can also be used to introduce DNA from outside the range of variation found in the cattle genome using a donor template with exogenous DNA sequences, in which case the resulting animal carries exogenous or “transgenic” DNA and is a genetically engineered animal (Figure 1). <br />
<center><br />
[[File:GE_Figure1.png]]<br />
</center><br />
'''Figure 1.''' Nuclease-induced double-strand breaks (DSBs) can be repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Imprecise NHEJ-mediated repair can produce insertion and/or deletion mutations of variable length at the site of the DSB. HDR-mediated repair can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template. Image from Sander and Joung, 2014<ref>Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355. doi:10.1038/nbt.2842.</ref>.<br />
<br />
Genome editing can be performed in cell culture followed by somatic cell nuclear transfer (SCNT) cloning in which case the genotype of the animal can be known for certain by genotyping or sequencing the cell line prior to cloning. Alternatively, editing can be undertaken in the developing embryo in which case the genotype of the animal will not be known until after it is born<ref name="BandE">Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. J Exp Biol 223 doi:10.1242/jeb.207159.</ref>. And in that case of an edited embryo the resulting calf may be mosaic – meaning that the cells within the animal have more than one genotype <ref name="BandE"></ref>.<br />
<br />
In the United States at the current time ALL genome edits in food animals – regardless of the nature of the intended alteration or edit – '''are regulated as new animal drugs by the FDA.''' This means that the animal and its products (milk and meat) are considered unapproved animal drugs and are therefore not salable. To obtain a new animal drug approval, or even to obtain permission for the animals to enter the food chain, extensive documentation is required. The following is what was required by the FDA for a “food use authorization” for genome edited cattle under an Investigational New Animal Drug (INAD) and their offspring in 2018:<br />
<br />
* Description of animals proposed to enter the food supply:<br />
* Species, class, and number of requested investigational animals to enter the food supply<br />
* Breeding strategy used to produce the offspring <br />
* The purpose of the genetic alteration and the intended function<br />
*Description of the genetic alteration (location in the genome, (impact on protein expression, if any,) intended sequence, etc.) and details of how the genomic alteration was achieved. <br />
* Comparison of the genetically altered sequence to the naturally occurring sequence (sequence alignment)<br />
* Whole Genome Sequencing (WGS) data for:<br />
<ol><br />
* the edited parental animal(s) (including information on which animal(s) the data were collected from and the relation of these animal(s) to the offspring) <br />
* the unedited cell line (control)<br />
* the offspring of the genome edited animal<br />
These data should confirm the alteration as well as validate that there are no unintended modifications to the genome that impact animal safety.<br />
</ol><br />
* Health records for the edited cattle and comparison to non-edited cattle<br />
* Provide a nutritional compositional analysis of edible muscle tissues of the investigational animals that will enter food supply including key nutrients. This should be compared with equivalent analysis of a conventional counterpart. Provide information on the methodology used and how the samples were obtained to conduct the meat analysis. Also, information regarding any statistical significance of any observed differences related to natural variation. This analysis may focus on muscle, but if available, can also include data on provide liver, kidney and fat.<br />
* The Codex Guideline (CAC/CL 68-2008) defines key nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates as nutrients or enzyme inhibitors as anti-nutrients) or minor compounds (minerals, vitamins).<br />
<br />
It is worth noting with regard to “off-target” edits or unintended modifications to the genome that study of whole genome sequence data from 2703 individual cattle in the 1000 Bull Genomes Project revealed more than 86.5 million differences (variants) between different breeds of cattle. These variants included 2.5 million insertions and deletions (indels) of one, or more, base pairs of DNA, and 84 million single nucleotide variants, where one of the four nucleotides making up DNA (A, C, G, T) had been changed to a different one<ref>Hayes, B.J., Daetwyler, H.D., 2019. 1000 Bull Genomes Project to Map Simple and Complex Genetic Traits in Cattle: Applications and Outcomes. Annu Rev Anim Biosci 7, 89-102. doi:10.1146/annurev-animal-020518-115024.</ref>. A small fraction of these mutations have been selected by breeders owing to their beneficial effects on traits of economic importance. None of these naturally-occurring variants are known to produce ill effects on the consumers of milk or beef products. In fact, every meal we have ever consumed is genetically distinct from every other meal in terms of genomic DNA sequences. Genetic variation per se does not pose a unique hazard as it relates to food safety, and it is in fact the fuel that drives animal breeding programs. If an animal that is otherwise healthy and exhibits just the phenotype that is intended by the edit (e.g. changed coat color), then it is hard to envisage a hazard associated with unintended genomic alterations that warrants full genome sequencing. In addition, there is no way to differentiate between naturally occurring genomic alterations and those unintended modifications introduced by editing<ref>Van Eenennaam, A.L., Wells, K.D., Murray, J.D., 2019. Proposed U.S. regulation of gene-edited food animals is not fit for purpose. NPJ Sci Food 3, 3. doi:10.1038/s41538-019-0035-y.</ref>.<br />
<br />
= Recommendations =<br />
According to the International Committee for Animal Recording Section 18 – Guidelines for Breed Associations<br />
* ''Breed Associations should check the rules of their countries with regard to allowing gene-edited animals in the herd book.''<br />
* ''If an animal has been gene edited it should be recorded against the animal when registered and should appear on the Zootechnical Certificate.''<br />
<br />
==Data Recording==<br />
''Given this background, edits in the genome of cattle will be referred to as “intended alterations” and it is recommended that the following records should be obtained:''<br />
<br />
* ''ID and pedigree of the animal that carries the intended alteration(s)<br />
* ''Description of the intended alteration (e.g. gene targeted, location in the genome, and intended alteration e.g. allele substitution or SNP change)<br />
* ''Details of how the intended alteration was achieved (e.g. editing of somatic cells followed by cloning, or introduction of editing regents into zygote and which editing method)<br />
* ''Timeline of when the edits were made and by which entity (e.g. company or laboratory)<br />
* ''Data confirming the intended alteration occurred (e.g. Sanger sequencing of targeted genomic region showing knockout or knock-in) showing that the edits that were actually made<br />
* ''If a donor template plasmid was used in the editing process data should be obtained to confirm the presence or absence of the editing regents in the genome (i.e. data as to whether there was integration of the plasmid backbone into the genome)<br />
* ''Data documenting whether the animal is heterozygous monollelic edit, a homozygous biallelic edit, a compound heterozygous biallelic edit, or mosaic for the intended alteration (See Figure 2)<br />
* ''The entity performing the editing should store the information they used to make the animal, and confirm the edits and should provide a certificate to the breeder/breed association confirming the genotype of the animal (e.g. homozygous biallelic polled (PP), heterozygous monoallelic slick (Ss)). <br />
* ''If an animal is mosaic it would be useful to know if it is germline mosaic as this can affect expected Mendelian inheritance ratios in the offspring. This can take some time as it requires the animal to sexually mature and either test semen or produce offspring, and so perhaps what is important is that breeders know the animal is mosaic so they are aware of this possibility.''<br />
<center><br />
[[File: GE_Figure2.png]]<br />
</center><br />
'''Figure 2.''' Schematic representation of possible outcomes from CRISPR-mediated mutation by cytoplasmic injection of an in vitro fertilized zygote (one cell embryo). 2n = number of homologous chromosomes, i.e. diploid. 2c/4c = number of copies of chromosomes either before or after DNA replication. Image from Supplementary materials of Hennig et al., 2020<ref>Hennig, S.L., Owen, J.R., Lin, J.C., Young, A.E., Ross, P.J., Van Eenennaam, A.L., Murray, J.D., 2020. Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos. Scientific Reports 10, 22309. doi:10.1038/s41598-020-78264-8.</ref>.<br />
=References=</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=File:GE_Figure2.png&diff=2450File:GE Figure2.png2021-09-18T15:10:01Z<p>AlisonVE: </p>
<hr />
<div></div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=File:GE_Figure1.png&diff=2449File:GE Figure1.png2021-09-18T14:52:45Z<p>AlisonVE: AlisonVE uploaded a new version of File:GE Figure1.png</p>
<hr />
<div></div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=File:GE_Figure1.png&diff=2448File:GE Figure1.png2021-09-18T14:49:40Z<p>AlisonVE: AlisonVE uploaded a new version of File:GE Figure1.png</p>
<hr />
<div></div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=File:GE_Figure1.png&diff=2447File:GE Figure1.png2021-09-18T14:44:29Z<p>AlisonVE: </p>
<hr />
<div></div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Genotyping&diff=297Genotyping2019-01-30T19:32:32Z<p>AlisonVE: </p>
<hr />
<div><br />
Genotyping refers to the process of using laboratory methods to determine which alleles an individual animal carries, usually at one particular gene or “locus” in the genome. The genotype identifies which alleles an animal carries. Producers must send in samples containing DNA from animals to be tested to the testing lab. Because all cells contain DNA, it is possible to genotype many different tissue types; however, laboratories may differ in their preferred sample type. Typical samples include blood vials or cards, semen, and tail hair samples. It is important that tail hair samples include the roots – ideally 30-50 hairs with intact roots. Below are links to videos on sampling DNA from cattle using different methods. <br />
<br />
If you are sampling DNA from a deceased animal call the testing laboratory to determine the best protocol. It is important to get a good quality sample to ensure the DNA test will be able to generate results. The cost of testing varies depending upon the company and how many tests are performed but ranges from $10-40/test; with an average of ~$25/test. Irrespective of carrier animals in its pedigree, an animal that has been tested and found to be a non-carrier did not inherit the mutant allele and will not transmit the genetic defect to its progeny.<br />
<br />
=Blood cards=<br />
<youtube>MSaY2o5CUck</youtube><br />
=Tail hair=<br />
<youtube>P3G-TYD5Uo8</youtube><br />
=TSU=<br />
<youtube>A9DX71M0uuU</youtube></div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Genotyping&diff=296Genotyping2019-01-30T19:24:42Z<p>AlisonVE: Created page with " Genotyping refers to the process of using laboratory methods to determine which alleles an individual animal carries, usually at one particular gene or “locus” in the gen..."</p>
<hr />
<div><br />
Genotyping refers to the process of using laboratory methods to determine which alleles an individual animal carries, usually at one particular gene or “locus” in the genome. The genotype identifies which alleles an animal carries. Producers must send in samples containing DNA from animals to be tested to the testing lab. Because all cells contain DNA, it is possible to genotype many different tissue types; however, laboratories may differ in their preferred sample type. Typical samples include blood vials or cards, semen, and tail hair samples. It is important that tail hair samples include the roots – ideally 30-50 hairs with intact roots. There are videos on sampling DNA from cattle tail hair ; and using an FTA blood card. If you are sampling DNA from a deceased animal call the testing laboratory to determine the best protocol. It is important to get a good quality sample to ensure the DNA test will be able to generate results. The cost of testing varies depending upon the company and how many tests are performed but ranges from $10-40/test; with an average of ~$25/test. Irrespective of carrier animals in its pedigree, an animal that has been tested and found to be a non-carrier did not inherit the mutant allele and will not transmit the genetic defect to its progeny.</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Monogenic_Traits&diff=295Monogenic Traits2019-01-30T19:18:34Z<p>AlisonVE: </p>
<hr />
<div>Monogenic traits – that is characteristics that are fully determined by one gene – tend to be the exception rather than the rule in livestock genetics. Having said that there are a number of important traits such as coat color and some genetic conditions that are monogenic and so understanding their inheritance important.<br />
<br />
All individuals receive one copy (allele) of each gene from their mother, and one from their father. The DNA sequence of a gene inherited from each parent may be identical in which case the individual is said to be homozygous for that gene, or the sequence of a gene inherited from each parent may vary in which case the individual is said to be heterozygous. Alleles can be recessive, meaning that an animal must inherit the same allele from both parents (i.e. be homozygous) before there is an effect, additive meaning that the effect is proportional to the number of alleles inherited by the animal (i.e. carrying two copies of a particular allele produces double the effect of carrying one copy), or dominant meaning that the inheritance of a single dominant allele can completely mask the expression of the allele inherited from the other parent.<br />
<br />
For simply inherited traits one allele completely masks the expression of the other allele when the alleles are heterozygous for the gene. This results in heterozygote animals having the exact same phenotype as homozygote dominant animals. This is the type of dominance we see in red/black coat color, where black is dominant to red. Cattle that have two black alleles are black (homozygous dominant), cattle that have one black and one red allele are also black (heterozygous), and red animals are the result of having two red alleles (homozygous recessive).<br />
<br />
When dealing with traits with complete dominance, heterozygous animals are often called carriers because they are carrying the recessive allele and can pass it to their offspring even though they do not express the recessive phenotype themselves. That is why it is possible to breed two black animals and get a red calf; each parent was a red allele carrier.<br />
<br />
Coat color is a good trait to demonstrate how alleles interact in a trait with complete dominance. For this example, we will mate an Angus bull to Hereford cows. The Angus bull is homozygous dominant, which means he has two black alleles (BB). The Hereford cows are homozygous recessive, which means they have two red alleles (bb). When mated, all offspring will be heterozygotes (Bb). The Punnett Square in Figure 1 illustrates this mating.<br />
<br />
'''Figure 1. Punnett Square for coat color when mating a homozygous black bull to homozygous red cows. The joining of the gametes shows the potential genotypes of offspring and their phenotype (color).'''<br />
<br />
[[File:Punnett_Square_Black_Bull_Red_Cow.png]]<br />
<br />
If we were to breed these heterozygous heifers back to a Hereford bull, we would get fifty percent heterozygous black (Bb) calves and fifty percent homozygous red (bb) calves (Figure 2).<br />
<br />
'''Figure 2. Punnett Square for coat color when mating a homozygous red bull to heterozygous black cows. The joining of the gametes shows the potential offspring and their color.<br />
'''<br />
<br />
[[File:Punnett_Square_Hetero_Bull_Hetero_Cow.png]]<br />
<br />
If we were to mate the Hereford x Angus heifers to Hereford x Angus bulls then we would get all three possibilities: homozygous black (BB), heterozygous black (Bb) and homozygous red (bb) (Figure 3). The ratio would be 25%:50%:25%, respectively. The phenotypic ratio would be 75%:25% black to red.<br />
<br />
'''Figure 3. Punnett Square for coat color when mating a heterozygous black bull to heterozygous black cows. The joining of the gametes shows the potential offspring and their color.'''<br />
<br />
[[File:Punnett_Square_Red_Bull_Hetero_Cow.png]]<br />
<br />
Traits controlled by one gene, with complete dominance, are easy to understand, but can cause problems because of the possibility of carriers of [[Recessive Genetic Defects | recessive genetic defects]]. For some traits, the only way to detect carriers is through progeny testing, which is costly and time consuming. However, with advancements in molecular technologies, carriers can be identified for many traits by [[Genotyping | conducting a DNA test]] on a tissue sample (See below).<br />
<br />
The simplest way to avoid having homozygous recessive calves is to always breed to a homozygous dominant bull. With this breeding strategy, even if you have the undesirable allele in your herd it will never be expressed because only homozygous dominant or carrier calves will be produced. This strategy can improve markets in the case of the red and horn allele, and increase reproductive rates when lethal alleles are involved.<br />
<br />
<br />
For more in-depth information you can access these fact sheets:<br />
<br />
[https://articles.extension.org/pages/74049/simple-inheritance-in-beef-cattle/ Simple Inheritance in Beef Cattle]<br />
<br />
[https://articles.extension.org/pages/72661/genetic-defects#/ Genetic Defects]<br />
<br />
[https://articles.extension.org/pages/72662/managing-genetic-defects#/ Managing Genetic Defects]<br />
<br />
[https://articles.extension.org/pages/73401/the-genetics-of-horned-polled-and-scurred-cattle/ The Genetics of Horned, Polled and Scurred Cattle]</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Recessive_Genetic_Defects&diff=294Recessive Genetic Defects2019-01-30T19:15:21Z<p>AlisonVE: Created page with "Many genetic defects are recessive, and the reason for this is that mutant alleles often render the resulting protein nonfunctional. In many cases if an individual inherits a..."</p>
<hr />
<div>Many genetic defects are recessive, and the reason for this is that mutant alleles often render the resulting protein nonfunctional. In many cases if an individual inherits a functioning allele of a gene from one parent, there is no phenotype associated with inheriting the nonfunctional mutant allele from the other parent. As such a heterozygous “Aa” animal, or carrier, appears normal. It is only when two carriers mate that they have the possibility of producing offspring that have by chance inherited both of the non-functional alleles from their parents. The example gene combinations that can occur with an autosomal recessive genetic condition are shown in Figure 4. Note that if this is a lethal genetic condition then all of the animals that are represented as solid black would not be alive and so the only possible matings would be between unaffected (green) and carrier (green and red) individuals.<br />
<br />
'''Figure 4. Mating combinations possible with an autosomal recessive genetic condition.'''<br />
[[File:Genetic_Condition_Mating.jpg]]<br />
<br />
All animals are carriers of mutations somewhere in their DNA for one or many recessive traits. Because an animal must inherit two copies of a given recessive mutation to be affected, and with only a few animals typically sharing the same mutation in the whole population, there is rarely a mating cross that has the potential to create affected offspring under natural selection. It is when relatives are mated that there is an increased possibility that offspring will inherit the mutant allele on both sides of the family tree. The Online Mendelian Inheritance in Animals (OMIA) is a catalogue/compendium of inherited disorders, other (single-locus) traits, and genes in 244 animal including an extended list of breed- defining characteristics, such as coat color, polledness, double- muscling and twinning. There are currently 523 total traits and disorders listed for cattle at this website. Table 1 lists the genetic conditions that are currently being monitored by U.S. breed associations<br />
<br />
'''Table I. Recessive genetic conditions currently being monitored by U.S. breed associations.'''<br />
<br />
{| class="wikitable"<br />
|-<br />
! Genetic Abnormality<br />
! Primary Breed(s) of Incidence<br />
! Lethal or Nonlethal<br />
! DNA Test Available<br />
|-<br />
| Alpha (α)-Mannosidosis (MA)<br />
| Red Angus<br />
| Lethal<br />
| Yes<br />
|-<br />
| Arthrogryposis Multiplex (AM)<br />
| Angus<br />
| Lethal<br />
| Yes<br />
|-<br />
| Beta (ß)-Mannosidosis<br />
| Salers<br />
| Lethal<br />
| Yes<br />
|-<br />
| Bovine Blood Coagulation Factor XIII Deficiency (F13)<br />
| Wagyu<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Chediak-Higashi Syndrome (CHS)<br />
| Wagyu<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Claudin 16 Deficiency (CL16)<br />
| Wagyu<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Contractural Arachnodactyly (CA)<br />
| Angus<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Developmental Duplication (DD)<br />
| Angus<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Dwawrfism (D2)<br />
| Angus<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Bulldog Dwarfism (BD)/ (Chondrodysplasia)<br />
| Dexter<br />
| Lethal<br />
| Yes<br />
|-<br />
| Erythrocyte Membrane Protein Band III Deficiency (Spherocytosis) (Band 3)<br />
| Wagyu<br />
| Often lethal<br />
| Yes<br />
|-<br />
| Hypotrichosis (hairless calf)<br />
|Hereford<br />
| Nonlethal<br />
| No<br />
|-<br />
| Factor XI Deficiency (F11)<br />
| Wagyu<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Freemartin (FM)<br />
| All<br />
| Sterile female<br />
| Yes<br />
|-<br />
| Idiopathic Epilepsy (IE)<br />
| Hereford<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Neuropathic Hydrocephalus (NH)<br />
| Angus<br />
| Lethal<br />
| Yes<br />
|-<br />
| Osteopetrosis (OS)<br />
| Angus and Red Angus<br />
| Lethal<br />
| Yes (Red Angus)<br />
|-<br />
| Protoporphyria<br />
| Limousin<br />
| Nonlethal<br />
| Yes<br />
|-<br />
| Pulmonary Hypoplasia and Anasarca (PHA)<br />
| Dexter, Maine-Anjou and Shorthorn<br />
| Lethal<br />
| Yes<br />
|-<br />
| Tibial Hemimelia (TH) <br />
| Shorthorn and Maine-Anjou<br />
| Lethal<br />
| Yes<br />
|}</div>AlisonVEhttp://guidelines.beefimprovement.org/index.php?title=Monogenic_Traits&diff=293Monogenic Traits2019-01-30T19:14:06Z<p>AlisonVE: Created page with "Monogenic traits – that is characteristics that are fully determined by one gene – tend to be the exception rather than the rule in livestock genetics. Having said that th..."</p>
<hr />
<div>Monogenic traits – that is characteristics that are fully determined by one gene – tend to be the exception rather than the rule in livestock genetics. Having said that there are a number of important traits such as coat color and some genetic conditions that are monogenic and so understanding their inheritance important.<br />
<br />
All individuals receive one copy (allele) of each gene from their mother, and one from their father. The DNA sequence of a gene inherited from each parent may be identical in which case the individual is said to be homozygous for that gene, or the sequence of a gene inherited from each parent may vary in which case the individual is said to be heterozygous. Alleles can be recessive, meaning that an animal must inherit the same allele from both parents (i.e. be homozygous) before there is an effect, additive meaning that the effect is proportional to the number of alleles inherited by the animal (i.e. carrying two copies of a particular allele produces double the effect of carrying one copy), or dominant meaning that the inheritance of a single dominant allele can completely mask the expression of the allele inherited from the other parent.<br />
<br />
For simply inherited traits one allele completely masks the expression of the other allele when the alleles are heterozygous for the gene. This results in heterozygote animals having the exact same phenotype as homozygote dominant animals. This is the type of dominance we see in red/black coat color, where black is dominant to red. Cattle that have two black alleles are black (homozygous dominant), cattle that have one black and one red allele are also black (heterozygous), and red animals are the result of having two red alleles (homozygous recessive).<br />
<br />
When dealing with traits with complete dominance, heterozygous animals are often called carriers because they are carrying the recessive allele and can pass it to their offspring even though they do not express the recessive phenotype themselves. That is why it is possible to breed two black animals and get a red calf; each parent was a red allele carrier.<br />
<br />
Coat color is a good trait to demonstrate how alleles interact in a trait with complete dominance. For this example, we will mate an Angus bull to Hereford cows. The Angus bull is homozygous dominant, which means he has two black alleles (BB). The Hereford cows are homozygous recessive, which means they have two red alleles (bb). When mated, all offspring will be heterozygotes (Bb). The Punnett Square in Figure 1 illustrates this mating.<br />
<br />
'''Figure 1. Punnett Square for coat color when mating a homozygous black bull to homozygous red cows. The joining of the gametes shows the potential genotypes of offspring and their phenotype (color).'''<br />
<br />
[[File:Punnett_Square_Black_Bull_Red_Cow.png]]<br />
<br />
If we were to breed these heterozygous heifers back to a Hereford bull, we would get fifty percent heterozygous black (Bb) calves and fifty percent homozygous red (bb) calves (Figure 2).<br />
<br />
'''Figure 2. Punnett Square for coat color when mating a homozygous red bull to heterozygous black cows. The joining of the gametes shows the potential offspring and their color.<br />
'''<br />
<br />
[[File:Punnett_Square_Hetero_Bull_Hetero_Cow.png]]<br />
<br />
If we were to mate the Hereford x Angus heifers to Hereford x Angus bulls then we would get all three possibilities: homozygous black (BB), heterozygous black (Bb) and homozygous red (bb) (Figure 3). The ratio would be 25%:50%:25%, respectively. The phenotypic ratio would be 75%:25% black to red.<br />
<br />
'''Figure 3. Punnett Square for coat color when mating a heterozygous black bull to heterozygous black cows. The joining of the gametes shows the potential offspring and their color.'''<br />
<br />
[[File:Punnett_Square_Red_Bull_Hetero_Cow.png]]<br />
<br />
Traits controlled by one gene, with complete dominance, are easy to understand, but can cause problems because of the possibility of carriers of [[Recessive Genetic Defects | recessive genetic defects]]. For some traits, the only way to detect carriers is through progeny testing, which is costly and time consuming. However, with advancements in molecular technologies, carriers can be identified for many traits by conducting a DNA test on a tissue sample (See below).<br />
<br />
<br />
Genotyping refers to the process of using laboratory methods to determine which alleles an individual animal carries, usually at one particular gene or “locus” in the genome. The genotype identifies which alleles an animal carries. Producers must send in samples containing DNA from animals to be tested to the testing lab. Because all cells contain DNA, it is possible to genotype many different tissue types; however, laboratories may differ in their preferred sample type. Typical samples include blood vials or cards, semen, and tail hair samples. It is important that tail hair samples include the roots – ideally 30-50 hairs with intact roots. There are videos on sampling DNA from cattle tail hair ; and using an FTA blood card. If you are sampling DNA from a deceased animal call the testing laboratory to determine the best protocol. It is important to get a good quality sample to ensure the DNA test will be able to generate results. The cost of testing varies depending upon the company and how many tests are performed but ranges from $10-40/test; with an average of ~$25/test. Irrespective of carrier animals in its pedigree, an animal that has been tested and found to be a non-carrier did not inherit the mutant allele and will not transmit the genetic defect to its progeny.<br />
<br />
For more in-depth information you can access these fact sheets:<br />
<br />
[https://articles.extension.org/pages/74049/simple-inheritance-in-beef-cattle/ Simple Inheritance in Beef Cattle]<br />
<br />
[https://articles.extension.org/pages/72661/genetic-defects#/ Genetic Defects]<br />
<br />
[https://articles.extension.org/pages/72662/managing-genetic-defects#/ Managing Genetic Defects]<br />
<br />
[https://articles.extension.org/pages/73401/the-genetics-of-horned-polled-and-scurred-cattle/ The Genetics of Horned, Polled and Scurred Cattle]</div>AlisonVE