CRISPR/Cas13系统敲减HEK293T细胞ku70和lig4基因表达

Knockdown the expression of ku70 and lig4 in HEK293T cells by CRISPR/Cas13 system

摘要:

成簇的规律间隔的短回文重复序列 (Clustered regularly interspaced short palindromic repeats, CRISPR)/CRISPR相关蛋白 (CRISPR-associated proteins，Cas) 系统是目前基因编辑、基因表达研究的热点，其中靶向RNA的CRISPR/Cas13系统的开发为RNA的干扰和编辑提供了新的方向。文中针对HEK293T细胞非同源末端连接 (Nonhomologous end joining，NHEJ) 通路修复关键因子Ku70和Lig4的编码序列，设计并合成CRISPR/Cas13a、b系统相应的gRNA，检测其对ku70和lig4基因表达的影响。结果显示，Cas13a对ku70和lig4的RNA敲减效果可以达到50%以上，Cas13b对ku70和lig4的RNA敲减效果分别达到92%和76%；同时Cas13a、b系统能将Ku70和Lig4蛋白水平分别下调至68%和53%。CRISPR/Cas13系统可有效敲减HEK293T细胞RNA和蛋白质表达，为基因功能和调控研究提供一种新的策略。

Abstract:

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.