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.

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.

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.

11Introducing useful traits into livestock breeding programs through gene knock-ins has 12 proven challenging. Typically, targeted insertions have been performed in cell lines, followed by 13 somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce 14 genome editing reagents and a homologous recombination (HR) donor template into embryos to 15 trigger homology-directed repair (HDR). However, the HR pathway is primarily restricted to 16 actively dividing cells (S/G2-phase) and its efficiency is low in zygotes, especially for the 17 introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based 18 strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved 19 knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted 20 when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor 21 template, or a HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly 22 higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). 23Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach 24 will facilitate the one-step introduction of gene constructs at a specific location of the bovine 25 genome and contribute to the next generation of elite cattle. 26 27Many attempts have been made to increase the rate of homologous recombination (HR) or 47 decrease the rate of non-homologous end joining (NHEJ) for gene insertion when using the 48 CRISPR/Cas9 system via CPI of zygotes 11 . However, these approaches have been unsuccessful in 49 bovine embryos as HR is primarily restricted to actively dividing cells 12 . Alternative homology 50 directed repair (HDR) approaches have been utilized for KI using a donor template via the 51 homology-mediated end joining (HMEJ) method 13 . This method has been shown to be active in 52 gametes and early stage 1-cell embryos, in which proteins necessary for pushing DNA repair 53 machinery towards the end-joining pathways are at their highest concentration 11 . While NHEJ 54 utilizes the Ku proteins and a ligase to mend the double-strand break (DSB) by blunt end ligation, 55 the HMEJ approach utilizes proteins for resection of the 5' ends and annealing of homologous 56 regions between the DSB and donor template similar to that in the microhomology-mediated end 57 joining (MMEJ) pathway 14 . 58In this study, we employed the HMEJ method for the precise insertion of the sex-59 determining region Y (SRY) gene into a region 10kb downstream of the zinc finger, X-linked (ZFX) 60 gene on the X chromosome of bovine embryos by injecting either in vitro matured oocytes prior 61 to in vitro fertilization, or presumptive zygotes 6hpi. We used two donor templates to compare KI 62 efficiency using the HMEJ and HR approaches, and show an increased rate of gene insertion at 63 the target location when using the HMEJ donor template. 64 RESULTS 65 Guide-RNA design and testing 66...

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.