山羊CSN2基因内含子7靶向的CRISPR-Cas9系统高效切割位点筛选

Highly efficient CRISPR-Cas9 system cleavage site selection in goat CSN2 intron7

【目的】使用CRISPR-Cas9系统在山羊CSN2基因座内含子7上筛选出高效的切割位点.【方法】根据"20N+NGG"原则在CSN2内含子7序列中设计了5个sgRNA靶向位点.分别连接具有sgRNA到和Cas9表达框的PX330-P2A-eGFP载体上,通过脂质体转染到实验室保存山羊胎儿成纤维细胞系中,PCR扩增转染阳性的细胞基因组CSN2内含子7序列,连接到Pmd19-T载体中,测序比较不同的sgRNA介导切割产生的突变.【结果】使用5个sgRNA均可以在山羊CSN2基因内含子7上造成有效切割,产生碱基删除或者插入突变.实验设计的5个sgRNA介导的CRISPR-Cas9系统效率均在30.7%以上,效率最高的2号位点(sgRNA2)介导的切割效率达到了61.5%.【结论】研究确定了CRISPR-Cas9系统在山羊CSN2基因内含子7内进行高效切割的sgRNA,为之后在该基因位点进行高效的非同源介导基因敲入奠定了基础.

[Objective] To screen highly effective CRISPR-Cas9 system cleavage sites in goat CSN2 gene intron7. [Method] Five sgRNAs were designed according to the ＂20N＋NGG＂ principle targeting the CSN2 gene intron7 sequence. Goat embryonic fibroblasts cells were transfected with vectors with Cas9 ex- pression cassette and different sgRNAs driven by U6 promoter. 48 h later, the intron7 sequences of the transfected cells were amplified and PCR products were ligated to Pmdlg-T vectors and subject to Sanger sequencing. [Result] All five sgRNAs rendered effective cleavage at the targeting sites, higher than 30. 7%. The most effective sgRNA,sgRNA2 rendered 61.5% insertions and deletions at the intron targe- ting site. The research here investigated five sgRNAs targeting the goat CSN2 intron7 and validated the most effective one. [Conclusion] The study shed light on the following non-HDR knock in research at the goat CSN2 gene locus.