基于CRISPR-Cas9定向编辑TRAC基因的研究

目的 针对人源TCRα C基因(TRAC)进行CRISPR/Cas9-gRNA的设计,并验证设计的gRNA的有效性及其脱靶率.方法 利用CRISPR网站(http://crispr.mit.edu/)提供的靶向编辑位点筛选工具,针对TCRα C区各设计出了3个编辑位点,并据此构建CRISPR-Cas9-TCR敲除质粒,将构建好的质粒转染293T细胞系,通过流式细胞术结合PCR产物的T-A克隆测序等方法验证各个gRNA序列的编辑效率.再根据所预测的潜在脱靶位点,设计相应的上下游引物进行PCR扩增后测序,检测其是否出现脱靶.结果 在3组针对TCRα C区的gRNA中,site2-gRNA的编辑效率最高,并且无脱靶现象.结论 针对人源TCRα C基因设计的site2-gRNA对TCRα C区具有很好的编辑效果,且无脱靶现象,为消除杂合TCR分子的产生和改善TCR修饰的T细胞(TCR-T)过继性免疫治疗奠定了基础.

Study on genome editing targeting TRAC using CRISPR-Cas9 system

Objective To design a CRISPR/Cas9-gRNA targeting human TCR alpha gene,and evaluate the editing effectiveness and the off-target loci of the gRNA candidates.Methods 3 spacer sequences of gRNA targeting TCR alpha C gene were designed with a CRISPR website (http://crispr.mit.edu/),and CRISPR-Cas9-TCR editing plasmids were respectively constructed based on pX458 plasmid.The recombinant plasmids were separately transfected into 293T cells.After three days of transfection the genomic DNA were extracted,and the fragments containing editing sites were amplified by PCR and subject to T-A cloning and sequencing to evaluate the editing efficiency of each gRNA.To investigate whether there is off-target cleavage mediated by gRNA,the DNA fragments containing the potential off-target sites predicted by online tools were amplified by PCR and subject to sequencing analysis.Results In three candidate gRNAs targeting TCR alpha C region,the site2-gRNA displayed the highest editing efficiency,and no off-target cleavage was found.Conclusion The site2-gRNA targeting human TCR alpha C gene shows good editing efficiency without off-target cleavage,which provides an important basis for eliminating the production of hybrid TCR in TCR gene-modified T cell (TCR-T) and improving the therapeutic effect of TCR-T.