利用CRISPR-Cas9系统建立Prmt1敲除的小鼠胚胎干细胞系

目的利用CRISPR-Cas9系统构建Prmt1敲除的E14小鼠胚胎干细胞系,并分析验证其敲除效果。方法利用第3代基因编辑技术CRISPR-Cas9系统,针对Prmt1第4、5、6、7外显子设计4条gRNAs,gRNA两两组合转染E14细胞,特异性的敲除E14细胞中的Prmt1,并在蛋白质水平,mRNA水平和基因组水平分别验证敲除效果。结果与对照组相比,两组敲除实验组都检测不到Prmt1蛋白质和mRNA的表达,基因组测序结果分析显示,两实验组中Prmt1的mRNA均出现无义突变,使其mRNA的翻译异常终止。同时,实时定量PCR结果显示,精氨酸甲基转移酶家族其他成员Prmt2,Carm1和Prmt6 mRNA表达不受影响。结论成功获得特异性敲除Prmt1的E14细胞系,为研究Prmt1在胚胎干细胞中的作用提供了重要工具。

Establishment of Prmt1 Knockout Mouse Embryonic Stem Cell Lines Using the CRISPR-Cas9 System.

Objective To establish and confirm Prmt1 knockout mouse embryonic stem cell lines. Methods Following instructions of CRISPR-Cas9 system manual, four guide RNAs targeting Prmt1 exon4, exon5, exon6 and exon7 were designed. The gRNA combinations were then transfected into E14 cells. Cell lines were selected and verified from protein, mRNA and genome levels. Results Compared with the control group, Prmt1 protein and mRNA were not detected in both knockout groups. Genome sequencing proved that nonsense mutations occurred in Prmt1 mRNA, which further led to the abnormal translation termination. Meanwhile, quantitative PCR results showed that expression of other members of protein arginine methyltransferases, Prmt2,Carm1 and Prmt6, were not affected. Conclusion Prmt1 knockout E14 cell lines were successfully established, which provided important tools for the study of Prmt1 in mouse embryonic stem cells.