ROBUST AND EFFICIENT ACTIVE GENETICS GENE CONVERSION IN THE RAT AND MOUSE

Abstract

The utility of Active Genetic (AG) gene conversion systems in rats and mice holds great promise for facilitating the production of complex strains harboring multiple humanizing genes. The practical application of such systems requires the identification of a robust, reusable, and highly efficient system. By characterizing twenty-eight different promoter and target site pairs we aimed to define the parameters needed to establish an efficient conversion system in male and female rats and mice. Using three specific meiosis prophase I active promoters to drive Cas9 expression. We studied several variables, including the number of Cas9 target sites, the distance between target sites, the cis versus trans configuration in linked pairs, and the effect of Cas9 copy number. In the rat, three of twelve tested configurations provided efficient AG gene conversion in the 22% - 67% range, and four others catalyzed AG in the 0.7-1% range. The rat Ddx4 (Vasa) promoter provides higher AG efficiency than the Sycp1 promoter. In mice, ten of sixteen tested configurations, using the Sycp1 and pSycp1 promoters, provided efficiency in the 0.3% - 3.2% range. In rats, Cas9 expression levels are remarkably well correlated with AG gene conversion efficiency. The rat cis rCyp3A1/rCyp3A2 locus was the most successful configuration, with gene conversion efficiencies of 0.7%-67%. This target site has a special property; the two Cas9 target sites are nearly perfectly homologous in the 100 bases around the gRNA target site. Our findings identify key parameters that improve AG efficiency, including the use of two Cas9 target sites, and efficient promoters that drive high levels of Cas9 expression that are correctly timed during gamete development. These findings also uncover the unexpected benefit of high homology at paired gRNA target sites to promote efficiency. We provide new data to guide future efforts to develop yet further improved AG systems.