中国肿瘤生物治疗杂志2017，Vol.24Issue(12)：1362-1369,8.DOI:10.3872/j.issn.1007-385x.2017.12.004
定向编辑CTLA4基因的向导RNA体外合成体系的构建及其编辑效率
Optimization of screening and in vitro construction of small guide RNA targeting CTLA4 gene
摘要
Abstract
Objective:To investigate the in vitro synthesis of sgRNA that specific editing CTLA4 (cytotoxic T-lymphocyte-associated protein 4) gene based on the principle of CRISPR/CAS9 gene editing technology.Methods:Firstly,we predicted sgRNAs for CTLA4 locus by CRISPR website,and three sgRNAs (sgRNA1,sgRNA2,sgRNA3) were designed.The recombinant sgRNA plasmids were constructed and transfected into 293T cells.After 48h,the genomic DNA of 293T cells was extracted,and the sgRNA sequence with high activity was screened with T7EN1 endonuclease.Subsequently,the screened sgRNA recombinant plasmid was selected as template to design the premier and further amplify the sgRNA core fragment using PCR technology;and then it was transcribed into sgRNA in vitro after optimizing the transcription conditions.Moreover,the cutting efficiency of purified sgRNA was assessed in vitro by Cas9 nuclease.And finally the sgRNA and the transcribed Cas9 mRNA were electroporated into CD3+ T cell of the lung cancer patients to knockout the target gene.Results:The T7EN1 endonuclease experiment verified that the knockout rate of sgRNA2 was the maximum with an efficiency up to 66％;after optimization of in vitro synthesis system,the activity and the yield of obtained CTLA4 sgRNA2 were high;it successfully cut the target double-stranded DNA at the designed site,and Cas9 nuclease detected the cutting efficiency of CTLA4 sgRNA2 reached 65％.Finally the CTLA4 gene can be effectively knocked out in CD3+ T cell of the lung cancer patients,and the amount of CTLA4 expression decreased from 46％ to 22％,the T7E1 endonuclease experiment also verified that the editing efficiency of sgRNA2 was 56％.Conclusion:A sgRNA with high editing efficiency targeting CTLA4 locus was obtained,which lays a foundation for further establish corresponding immunotherapy strategy targeting CTLA4 gene.关键词
肺癌/免疫检查点/CTLA4/CRISPR/Cas9/向导RNA/体外转录Key words
lung cancer/immune checkpoint/CTLA4/CRISPR/Cas9/sgRNA/in vitro transcription分类
医药卫生引用本文复制引用
任飞飞,李峰,李砺锋,赵启泰,黄岚,张毅..定向编辑CTLA4基因的向导RNA体外合成体系的构建及其编辑效率[J].中国肿瘤生物治疗杂志,2017,24(12):1362-1369,8.基金项目
国家重点研发计划重点专项资助(No.2016YFC1303501) （No.2016YFC1303501）
河南省医学科技攻关计划项目(No.201501004) （No.201501004）
河南省重大科技专项项目(No.1611003101000) （No.1611003101000）
郑州大学第一附属医院-中国科学院大连化学物理研究所合作项目.Project supported by the Research Grant from the National Key Research and Development Program of China (No.2016YFC1303501),the Medical Science and Technique Foundation from Ministry of Public Health of Henan Province (No.201501004),the Major Science and Technology Projects of Henan Province (No.1611003101000),and the Cooperative Project of the First Affiliated Hospital of Zhengzhou University and Dalian Institute of Chemical Physics of Chinese Academy of Sciences （No.2016YFC1303501）
