靶向切割猪Y染色体的CRISPR/Cas9载体构建及功能验证

【目的】通过CRISPR/Cas9系统对Y染色体多位点切割，实现目标染色体的敲除，为畜禽性别控制提供新的手段。【方法】以CRISPR/Cas9技术为基础，寻找Y染色体上能被sgRNA特异性识别的多拷贝重复序列，并通过体外切割、定量分析、核型鉴定验证其对靶点的有效性。【结果】所设计的sgRNA能够在体外实现对靶片段的明显切割，且切割效率均达到了50%以上。基因的定量分析结果证明了其在细胞水平切割的有效性，且簇状重复序列切割效果明显优于散在重复序列；核型鉴定结果证实了细胞水平猪Y染色体的丢失。【结论】研究结果为后续构建染色体敲除猪，实现猪的性别控制奠定了基础。

Construction and functional validation of CRISPR/Cas9 vector targeting pig Y chromosome cutting

Objective To knock out the target chromosome by CRISPR/Cas9 system to cut multiple sites of Y chromosome, and provide a new method for sex control of livestock and poultry. Method Based on CRISPR/Cas9 technology, we searched for multiple copies of repeat sequences on Y chromosome that can be specifically recognized by sgRNA, and verified their effectiveness on target by in vitro cleavage, quantitative analysis and karyotype identification. Result The designed sgRNA could cut the target fragment obviously in vitro, and the cutting efficiency was more than 50%. The results of quantitative analysis of genes further proved the effectiveness of gene cutting at the cell level, and the cutting effect of clustered repeats was significantly better than that of scattered repeats. Karyotype identification also confirmed the loss of pig Y chromosome at the cellular level. Conclusion The research results lay a foundation for the subsequent construction of chromosome knockout pigs and the realization of sex control in pigs.