Harnessing Endogenous Repair Mechanisms for Precise Modification of in Vitrofertilized Bovine Embryos

The use of genetic editing tools, such as the CRISPR/Cas9 system, could be used to introduce useful health and welfare characteristics such as disease resistance into cattle breeding programs. However, current methods for harnessing these tools for knock-ins or whole gene insertions have been limited to alterations in cell culture followed by somatic cell nuclear transfer (SCNT) cloning. This approach is problematic as SCNT cloning is inefficient, and edits are made in existing germplasm, rather than the next generation. One alternative is CRISPR-mediated modification of in vitrofertilized embryos by direct cytoplasmic injection. Overall, this method has been unsuccessful at achieving knock-ins in cattle embryos due to the low activity of the homologous recombination (HR) pathway in gametes and one-cell zygotes. Previous approaches for attempting gene knock-ins using homology directed repair (HDR) have primarily focused on improving HR function by knocking down the non-homologous end-joining (NHEJ) pathway, or attempting to enhance HR activity. These methods have been largely unsuccessful in embryos as double strand breaks are primarily resolved using the NHEJ pathway. We have developed an approach which utilizes the homology mediated end-joining (MHEJ) pathway to facilitate HDR via direct cytoplasmic injection of in vitro matured bovine oocytes prior to fertilization. Using this method we showed a significantly higher rate of gene knock-in as compared to approaches which utilize the HR pathway (34.0% vs. 0%; p < 0.0001). Along with the increased rate of gene integration at the target site, we observed a high level of non-mosaic embryos when using this approach (24.6%). As the end-joining pathway is the primary repair mechanism in gametes, this method harnesses the ability for HDR by direct injection of oocytes, utilizing the MHEJ pathway for targeted gene knock-in of embryos. This approach could enable precise germline editing of the next generation of elite bovine embryos.