CRISPR/Cas9-mediated knock-in of the murine Y chromosomal Sry gene.

Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flag). In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.