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1362~1369.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].任飞飞,李峰,李砺锋,赵启泰,黄岚,张毅.中国肿瘤生物治疗杂志,2017,24(12)
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    点此下载全文    点此浏览HTML全文
任飞飞  李峰  李砺锋  赵启泰  黄岚  张毅
1.郑州大学第一附属医院 生物细胞治疗中心,河南 郑州 450052;2. 郑州大学生命科学学院,河南郑州450052;3. 郑州大学第一附属医院肿瘤科,河南郑州450052,1.郑州大学第一附属医院 生物细胞治疗中心,河南 郑州 450052;2. 郑州大学生命科学学院,河南郑州450052;3. 郑州大学第一附属医院肿瘤科,河南郑州450052,1.郑州大学第一附属医院 生物细胞治疗中心,河南 郑州 450052;2. 郑州大学生命科学学院,河南郑州450052;3. 郑州大学第一附属医院肿瘤科,河南郑州450052,1.郑州大学第一附属医院 生物细胞治疗中心,河南 郑州 450052;2. 郑州大学生命科学学院,河南郑州450052;3. 郑州大学第一附属医院肿瘤科,河南郑州450052,1.郑州大学第一附属医院 生物细胞治疗中心,河南 郑州 450052;2. 郑州大学生命科学学院,河南郑州450052;3. 郑州大学第一附属医院肿瘤科,河南郑州450052,1.郑州大学第一附属医院 生物细胞治疗中心,河南 郑州 450052;2. 郑州大学生命科学学院,河南郑州450052;3. 郑州大学第一附属医院肿瘤科,河南郑州450052
基金项目:国家重点研发计划重点专项资助(No. 2016YFC1303501);河南省医学科技攻关计划项目(No. 201501004);河南省重大科技专项项目(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
DOI:10.3872/j.issn.1007-385X.2017.12.004
摘要:
      目的:基于CRISPR/Cas9 基因编辑技术原理体外合成定向编辑细胞毒性T 淋巴细胞相关抗原4 (cytotoxic T-lymphocyte-associated protein 4, CTLA4)基因的向导RNA(small guide RNA,sgRNA)。方法:(1)使用Crispr 网站对CTLA4 位点的sgRNA进行预测并设计出3 个sgRNA(sgRNA1、sgRNA2、sgRNA3),构建sgRNA 重组质粒,将其转染293T 细胞,48 h 后提取基因组DNA,利用T7EN1 核酸内切酶筛选活性较高的sgRNA序列;(2)以筛选到的sgRNA重组质粒为模板,设计引物并用PCR扩增体外合成sgRNA的DNA模板片段,进一步优化体外转录条件,转录出sgRNA,并利用Cas9 核酸酶体外检测转录纯化sgRNA的切割效率;(3)将sgRNA与转录得到的Cas9 mRNA 电转化入肺癌患者CD3+T细胞,实现靶基因敲除。结果:T7EN1 核酸内切酶实验证实了sgRNA2 序列靶向敲除效率最高,效率达到66%;优化体外合成体系后,转录得到的CTLA4 sgRNA2 产量高,具有较高的活性,在靶点上成功切开了DNA双链,Cas9 核酸酶检测出其切割效率达到了65%;最终可在肺癌患者CD3+T细胞中有效敲除CTLA4 基因,CTLA4 分子的表达从46%降低到22%,T7E1 核酸内切酶实验进一步证实了其编辑效率为56%。结论:体外成功构建了具有较高编辑效率的定向编辑CTLA4基因的sgRNA,为后续针对该基因制定相应的免疫治疗策略奠定了基础。
关键词:肺癌  免疫检查点  CTLA4  CRISPR/Cas9  向导RNA  体外转录
Optimization of screening and in vitro construction of small guide RNA targeting CTLA4 gene    Download Fulltext
REN Feifei  LI Feng  LI Lifeng  ZHAO Qitai  HUANG Lan  ZHANG Yi
1.Biotherapy Center, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; 2. School of Life Science, Zhengzhou University,Zhengzhou 450001, Henan, China; 3. Department of Oncology, First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052, Henan, China,1.Biotherapy Center, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; 2. School of Life Science, Zhengzhou University,Zhengzhou 450001, Henan, China; 3. Department of Oncology, First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052, Henan, China,1.Biotherapy Center, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; 2. School of Life Science, Zhengzhou University,Zhengzhou 450001, Henan, China; 3. Department of Oncology, First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052, Henan, China,1.Biotherapy Center, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; 2. School of Life Science, Zhengzhou University,Zhengzhou 450001, Henan, China; 3. Department of Oncology, First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052, Henan, China,1.Biotherapy Center, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; 2. School of Life Science, Zhengzhou University,Zhengzhou 450001, Henan, China; 3. Department of Oncology, First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052, Henan, China and 1.Biotherapy Center, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China; 2. School of Life Science, Zhengzhou University,Zhengzhou 450001, Henan, China; 3. Department of Oncology, First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052, Henan, China
Fund Project: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
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 wa s 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 experi-ment 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.
Keywords:lung cancer  immune checkpoint  CTLA4  CRISPR/Cas9  sgRNA  in vitro transcription
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