基于CRISPR/Cas9系统构建绵羊VASA基因敲入载体及验证

本研究旨在探索VASA基因在绵羊睾丸发育中的表达变化，并通过构建VASA基因敲入载体，为下一步进行绵羊生殖细胞体外诱导分化研究提供基础。采集性成熟前后即3月龄（3-month-old，3M）和9月龄（9-month-old，9M）绵羊睾丸组织，利用实时荧光定量PCR （quantitative real-time PCR，qPCR）和Western blotting技术分析VASA基因的差异表达，并利用免疫组织化学技术对VASA基因的表达定位进行分析。设计靶向VASA基因的向导RNA （guide RNA，gRNA），并构建同源重组载体，进行质粒转染绵羊耳成纤维细胞。结合CRISPR/dCas9技术对VASA基因进行激活，进一步验证载体效率。结果表明，VASA基因随着绵羊睾丸发育，表达水平极显著增加（P<0.01），且主要定位在精母细胞和圆形精子细胞中。利用CRISPR/Cas9系统构建了VASA基因敲入载体，联合pEGFP-PGK puro-VASA载体转染耳成纤维细胞，CRISPR/dCas9系统激活后，耳成纤维细胞成功表达VASA基因。结果提示，VASA基因在绵羊睾丸发育和精子发生中发挥潜在功能，且通过CRISPR/Cas9系统可在体外构建VASA基因敲入载体，为下一步探究VASA基因对绵羊雄性生殖细胞的发育和分化提供有效的研究手段。

Construction and validation of sheep VASA gene knock-in vector based on CRISPR/Cas9 system

This study aimed to explore the expression changes of VASA gene in sheep testis development and to construct VASA gene knock-in vector to prepare for the study on the differentiation of sheep germ cells in vitro. The testicular tissues of 3-month-old (3M) and 9-month-old (9M) sheep which represent immature and mature stages, respectively, were collected. The differential expression of VASA gene was analyzed by quantitative real-time PCR (qPCR) and Western blotting, and the location of VASA gene was detected by immunohistochemistry. The sgRNA targeting the VASA gene was designed and homologous recombination vectors were constructed by PCR. Subsequently, plasmids were transferred into sheep ear fibroblasts. The VASA gene was activated in combination with CRISPR/dCas9 technology to further verify the efficiency of the vector. The results showed that the expression level of VASA gene increased significantly with the development of sheep testis (P<0.01), and was mainly located in spermatocytes and round spermatids. The knock-in vector of VASA gene was constructed by CRISPR/Cas9 system, and the Cas9-gRNA vector and pEGFP-PGK puro-VASA vector were transfected into ear fibroblasts. After CRISPR/dCas9 system was activated, ear fibroblasts successfully expressed VASA gene. The results suggest that VASA gene plays a potential function in sheep testicular development and spermatogenesis, and the VASA gene knock-in vector can be constructed in vitro through the CRISPR/Cas9 system. Our results provided effective research tools for further research of germ cell development and differentiation.