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(Nonhomologousendjoining,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。
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/Cas13 a and CRISPR/Cas13 b systems in non-homologous end joining(NHEJ)pathway,such as Ku70 and Lig4,and then detected the expression of ku70 and lig4 in HEK293 T cells.The CRISPR/Cas13 a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%,and CRISPR/Cas13 b system also suppressed ku70 and lig4 about 92%and 76%,respectively.Also,CRISPR/Cas13 a,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 HEK293 T cells,providing a new strategy for gene function and regulation research.
作者
王豪强
李国玲
黄广燕
李紫聪
郑恩琴
徐铮
杨化强
吴珍芳
张献伟
刘德武
Haoqiang Wang;Guoling Li;Guangyan Huang;Zicong Li;Enqin Zheng;Zheng Xu;Huaqiang Yang;Zhenfang Wu;Xianwei Zhang;Dewu Liu(National Engineering Research Center for Breeding Swine Industry,College of Animal Science,South China Agricultural University,Guangzhou 510642,Guangdong,China;Wens Foodstuff Group Co.,Ltd,Yunfu 527400,Guangdong,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2020年第7期1414-1421,共8页
Chinese Journal of Biotechnology
基金
国家转基因重大专项(No.2016ZX08006002)
广东省科技创新战略专项(No.2018B020203002)资助。
