靶向敲除绒山羊lnc15479的CRISPR/Cas9构建及活性验证

Construction and Active Verification of lnc15479 Targeted Knockout System on Cashmere Goat by CRISPR/Cas9 Technology

为研究生长期和休止期lnc15479的差异表达在陕北白绒山羊毛囊周期性发育中的作用及功能,利用CRISPR/Cas9技术,在lnc15479的上、下游各设计1个sgRNA靶位点,构建CRISPR/cas9表达载体;并基于SSA修复机制,分别构建含有红色和绿色荧光标记的双荧光报告载体系统。通过将表达载体和报告载体共同转染HEK293T细胞,检测该CRISPR/Cas9系统的工作效率。结果表明,lnc15479的CRISPR/Cas9表达载体成功构建,根据红色和绿色荧光表达情况及细胞计数的方法,该系统的工作效率约为20%。研究结果为进一步分析lnc15479的功能提供技术支持。

In order to understand the function of Inc15479 in hair follicle periodic development, two sgRNA target sites in the upstream and downstream of Inc15479 were designed and the CRISPR/Cas9 expression vector was constructed. The double fluorescent reporter vector based on SSA repair mechanism was then constructed to detect the cleavage efficiency of CRISPR-cas9. The expression and re- port vectors were then co-transfected HEK293T cells to detect the work efficiency of CRISPR/Cas9. The results showed that CRISPR/Cas9 vector was successfully constructed and its cleavage efficiency was about 20% from the red and green fluoresence. The results would provide technical support for the further study on Inc15479.