Imaimatsu Kenya, Fujii Wataru, Hiramatsu Ryuji, Miura Kento, Kurohmaru Masamichi, Kanai Yoshiakira
Department of Veterinary Anatomy, The University of Tokyo, Tokyo 113-8657, Japan.
Department of Animal Resource Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
J Reprod Dev. 2018 Jun 22;64(3):283-287. doi: 10.1262/jrd.2017-161. Epub 2018 Apr 14.
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 (Y). In the F1 and successive generations, all male pups carrying the Y 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.
通过成簇规律间隔短回文重复序列/CRISPR相关核酸内切酶9（CRISPR/Cas9）系统进行的哺乳动物合子介导的基因组编辑被广泛用于生成基因组修饰动物。该系统可在小鼠中产生包括Sry在内的各种Y染色体基因的功能丧失突变。在此，我们报告了通过CRISPR-Cas9介导在Y染色体（Y）的C端编码末端的Sry基因座中敲入Flag标签序列品系的建立。在F1代及后续世代中，所有携带Y染色体的雄性幼崽在成熟时睾丸分化正常且精子发生正常，能够完全生育并产生可存活的后代。据我们所知，本研究首次在C端区域产生了稳定的Sry敲入品系，突出了一种用于研究哺乳动物原始Sry基因座处氨基酸变化意义的新方法。
