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

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