Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes.

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

Review: Progress in producing chimeric ungulate livestock for agricultural applications.

The progress made in recent years in the derivation and culture of pluripotent stem cells from farm animals opens up the possibility of creating livestock chimeras. Chimeras producing gametes exclusively derived from elite donor stem cells could pass superior genetics on to the next generation and thereby reduce the genetic lag that typically exists between the elite breeding sector and the commercial production sector, especially for industries like beef and sheep where genetics is commonly disseminated through natural service mating. Chimeras carrying germ cells generated from genome-edited or genetically engineered pluripotent stem cells could further disseminate useful genomic alterations such as climate adaptation, animal welfare improvements, the repair of deleterious genetic conditions, and/or the elimination of undesired traits such as disease susceptibility to the next generation. Despite the successful production of chimeras with germ cells generated from pluripotent donor stem cells injected into preimplantation-stage blastocysts in model species, there are no documented cases of this occurring in livestock. Here, we review the literature on the derivation of pluripotent stem cells from ungulates, and progress in the production of chimeric ungulate livestock for agricultural applications, drawing on insights from studies done in model species, and discuss future possibilities of this fast-moving and developing field. Aside from the technical aspects, the consistency of the regulatory approach taken by different jurisdictions towards chimeric ungulate livestock with germ cells generated from pluripotent stem cells and their progeny will be an important determinant of breeding industry uptake and adoption in animal agriculture.

Field demonstration analyzing the implementation of individual animal electronic identification and genetic testing in western range sheep flocks.

Adoption of electronic identification ear tags (EID) and DNA testing by commercial range sheep producers in the Western United States has been low, despite the availability of these technologies for over a decade. Jointly, these technologies offer an approach to provide individual animal performance data to improve flock health, genetic and reproductive management. This project involved a collaboration with five California sheep producers representing a broad geographic range, varying levels of pre-project EID adoption, and diverse operational practices. Tissue samples were collected from, and ear EIDs were placed in, a total of 2,936 rams and their potential lambs. We partnered with a commercial packing company, Superior Farms, to genotype the animals. Superior Farms used a targeted genotyping panel to assign parentage, and link individual animal identification (ID) to camera-graded carcass measurements. This enabled the collection of individual progeny carcass data and provided insight into sire performance, providing for the within-flock identification of prolific sires that were producing lambs with significantly more saleable meat as compared to their flock mates. Overall, almost 91% of lambs were successfully matched to their sire, and prolificacy ranging from 0-135 lambs per ram. There was as much as an $80 difference in the average edible product from camera-graded carcasses derived from lamb groups sired by different rams. A partial budget analysis modeling investment in an EID system coupled with an autodrafter and scale to collect individual weights and improve labor efficiency during processing, and a sheep flip chute to improve worker safety during foot trimmings, yielded a greater than 7:1 return on investment over a five-year time frame. Ideally, the data collection enabled by EIDs and DNA testing would feed into data-driven genetic evaluation programs to enable selection for more productive and profitable animals, and allow the US sheep industry to accelerate the rate of genetic improvement.

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele.

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5' and the 3' regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant PC POLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (PCp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.

A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle.

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

Etiology and risk factors for bovine respiratory disease in pre-weaned calves on California dairies and calf ranches.

Our study objective was to estimate the magnitude of association of BRD risk factors including failure of passive immunity transfer, sex, age, and the detection of suspected BRD etiological pathogens in pre-weaned dairy calves in California. A conditional logistic regression model and a mixed-effects logistic regression model were used to estimate the association of these potential risk factors with BRD from a matched and nested case-control studies, respectively. For each exposure covariate, the odds ratio (OR) is the ratio of odds of an exposure in a BRD calf (case) to that in a non-BRD calf (control). In the matched case-control study, an interaction term between failure of transfer of passive immunity and sex of calf showed that female calves were more negatively impacted by failure of transfer of passive immunity compared to male calves. The odds ratios comparing failure of transfer of passive immunity in BRD score positive calves versus controls for male calves was 1.34 (95 % CI: 0.87, 2.06) and was 2.47 (95 % CI: 1.54, 3.96) for female calves. The model odds ratios varied from 1.74 (95 % CI: 1.26, 2.42) for Mycoplasma spp. to 9.18 (95 % CI: 2.60, 32.40) for Histophilus somni, with Mannheimia haemolytica and Pasteurella multocida having an OR of 6.64 (95 % CI: 4.39, 10.03) and 6.53 (95 % CI: 4.44, 9.59), respectively. For bovine respiratory syncytial virus positive calves, the OR was 4.60 (95 % CI: 3.04, 6.97). Findings from the nested case-control study showed that based on thoracic ultrasonography findings consistent with BRD, the odds of a calf being 1 day older compared to a day younger were 1.01 (95 % CI: 1.00, 1.02) among BRD cases. For the bacterial and viral pathogens, the OR for Mycoplasma spp. and Pasteurella multocida were 1.85 (95 % CI: 1.24, 2.75) and 1.86 (95 % CI: 1.28, 2.71), respectively. The OR values for these pathogens were similar when both thoracic auscultation and ultrasound findings were used to detect cases of BRD. Based on positive scores for BRD using the California BRD scoring system, the OR for facility type, calf ranch versus dairy farm, was 3.17 (95 % CI: 1.43, 7.01), Mannheimia haemolytica was 3.50 (95 % CI: 2.00, 6.11), Pasteurella multocida was 1.78 (95 % CI: 1.21, 2.60), and bovine coronavirus was 2.61 (95 % CI: 1.85, 3.70). Results from both study designs showed the difference in relative contributions of age, sex, immune status, and pathogens in BRD occurrence between cases and controls in pre-weaned dairy calves.

Electroporation-Mediated Genome Editing of Livestock Zygotes.

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

Functional annotations of three domestic animal genomes provide vital resources for comparative and agricultural research.

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources.

Comparison of Gene Editing Versus Conventional Breeding to Introgress the POLLED Allele Into the Tropically Adapted Australian Beef Cattle Population.

Dehorning is the process of physically removing horns to protect animals and humans from injury, but the process is costly, unpleasant, and faces increasing public scrutiny. Genetic selection for polled (hornless), which is genetically dominant to horned, is a long-term solution to eliminate the need for dehorning. However, due to the limited number of polled Australian Brahman bulls, the northern Australian beef cattle population remains predominantly horned. The potential to use gene editing to produce high-genetic-merit polled cattle was recently demonstrated. To further explore the concept, this study simulated introgression of the POLLED allele into a tropically adapted Australian beef cattle population via conventional breeding or gene editing (top 1% or 10% of seedstock bulls/year) for 3 polled mating schemes and compared results to baseline selection on genetic merit (Japan Ox selection index, $JapOx) alone, over the course of 20 years. The baseline scenario did not significantly decrease the 20-year HORNED allele frequency (80%), but resulted in one of the fastest rates of genetic gain ($8.00/year). Compared to the baseline, the conventional breeding scenarios where polled bulls were preferentially used for breeding, regardless of their genetic merit, significantly decreased the 20-year HORNED allele frequency (30%), but resulted in a significantly slower rate of genetic gain ($6.70/year, P ≤ 0.05). The mating scheme that required the exclusive use of homozygous polled bulls, resulted in the lowest 20-year HORNED allele frequency (8%), but this conventional breeding scenario resulted in the slowest rate of genetic gain ($5.50/year). The addition of gene editing the top 1% or 10% of seedstock bull calves/year to each conventional breeding scenario resulted in significantly faster rates of genetic gain (up to $8.10/year, P ≤ 0.05). Overall, our study demonstrates that, due to the limited number of polled Australian Brahman bulls, strong selection pressure on polled will be necessary to meaningfully increase the number of polled animals in this population. Moreover, these scenarios illustrate how gene editing could be a tool for accelerating the development of high-genetic-merit homozygous polled sires to mitigate the current trade-off of slower genetic gain associated with decreasing HORNED allele frequency in the Australian Brahman population.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

BACKGROUND: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. RESULTS: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. CONCLUSION: The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route. Additionally, the costs associated with delayed commercialization of GE livestock were modeled using three case studies: GE mastitis-resistant dairy cattle, genome-edited porcine reproductive and respiratory syndrome virus-resistant pigs, and the AquAdvantage salmon. Delays of 5 or 10 years in the commercialization of GE livestock beyond the normative 10-year GE product evaluation period were associated with billions of dollars in opportunity costs and reduced global food security.

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos.

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos.

Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.

A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues.

BACKGROUND: Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues. RESULTS: Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions. CONCLUSIONS: The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

Genome editing approaches to augment livestock breeding programs.

The prospect of genome editing offers a number of promising opportunities for livestock breeders. Firstly, these tools can be used in functional genomics to elucidate gene function, and identify causal variants underlying monogenic traits. Secondly, they can be used to precisely introduce useful genetic variation into structured livestock breeding programs. Such variation may include repair of genetic defects, the inactivation of undesired genes, and the moving of useful alleles and haplotypes between breeds in the absence of linkage drag. Editing could also be used to accelerate the rate of genetic progress by enabling the replacement of the germ cell lineage of commercial breeding animals with cells derived from genetically elite lines. In the future, editing may also provide a useful complement to evolving approaches to decrease the length of the generation interval through in vitro generation of gametes. For editing to be adopted, it will need to seamlessly integrate with livestock breeding schemes. This will likely involve introducing edits into multiple elite animals to avoid genetic bottlenecks. It will also require editing of different breeds and lines to maintain genetic diversity, and enable structured cross-breeding. This requirement is at odds with the process-based trigger and event-based regulatory approach that has been proposed for the products of genome editing by several countries. In the absence of regulatory harmony, researchers in some countries will have the ability to use genome editing in food animals, while others will not, resulting in disparate access to these tools, and ultimately the potential for global trade disruptions.

Author Correction: Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull.

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around 'intentional genomic alterations' and future regulatory actions regarding genome-edited animals.

Management of lethal recessive alleles in beef cattle through the use of mate selection software.

BACKGROUND: Recessive loss-of-function (LOF) alleles at genes which are essential for life, can result in early embryonic mortality. Cattle producers can use the LOF carrier status of individual animals to make selection and mate allocation decisions. METHODS: Two beef cattle breeding strategies i.e. (1) selection against LOF carriers as parents and (2) simultaneous selection and mate allocation to avoid the occurrence of homozygous offspring in three scenarios, which differed in number and frequency of LOF alleles were evaluated using the mate selection program, MateSel. Scenarios included (a) seven loci with high-frequency LOF alleles, (b) 76 loci with low-frequency LOF alleles, and (c) 50 loci with random high- and low-frequency LOF alleles. In addition, any savings resulting from the information obtained by varying the percentage (0-100%) of the herd genotyped, together with segregation analysis to cover ungenotyped animals, were calculated to determine (1) which percentage optimized net profit for a fixed cost of genotyping ($30/test), and (2) the breakeven cost for genotyping. RESULTS: With full knowledge of the LOF alleles carried by selection candidates, the most profitable breeding strategy was always simultaneous selection and mate allocation to avoid homozygous affected offspring (aa) as compared to indiscriminate selection against carrier parents (Aa). The breakeven value of genotyping depended on the number of loci modeled, the LOF allele frequencies, and the mating/selection strategies used. Genotyping was most valuable when it was used to avoid otherwise high levels of embryonic mortalities. As the number of essential loci with LOF alleles increased, especially when some were present at relatively high minor allele frequencies, embryonic losses increased, and profit was maximized by genotyping 10 to 20% of a herd and using that information to reduce these losses. CONCLUSIONS: Genotyping 100% of the herd was never the most profitable outcome in any scenario; however, genotyping some proportion of the herd, together with segregation analysis to cover ungenotyped animals, maximized overall profit in scenarios with large numbers of loci with LOF alleles. As more LOF alleles are identified, such a mate selection software will likely be required to optimally select and allocate matings to balance the rate of genetic gain, embryonic losses, and inbreeding.