PT  - JOURNAL ARTICLE
AU  - Joseph R. Owen
AU  - Sadie L. Hennig
AU  - Bret R. McNabb
AU  - Jason C. Lin
AU  - Amy E. Young
AU  - James D. Murray
AU  - Pablo J. Ross
AU  - Alison L. Van Eenennaam
TI  - Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos
AID  - 10.1101/2020.06.09.143115
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.06.09.143115
4099  - http://biorxiv.org/content/early/2020/06/12/2020.06.09.143115.short
4100  - http://biorxiv.org/content/early/2020/06/12/2020.06.09.143115.full
AB  - 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 is low in zygotes, especially for the introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor template, or a 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.Competing Interest StatementThe authors have declared no competing interest.