Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair.

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion. [Display omitted] • Interhomolog repair (IHR) occurs throughout the genome in the early mouse embryo • RAD51, BCCIP, and USP1/WDR48 enhance IHR at sites of Cas9-induced DNA breaks • Embryonic IHR occurs via gene conversion without crossover • Enhanced IHR can be used for homozygous knockin and donor-free allelic conversion Co-injection of mouse zygotes with RAD51 and Cas9 can enhance interhomolog repair and facilitate conversion of heterozygous alleles to homozygous alleles. [ABSTRACT FROM AUTHOR]