CD8~+ T细胞PD-1基因CRISPR/Cas9编辑体系电穿孔条件的优化
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摘要
目的:通过优化电穿孔递送条件,提高基于CRISPR/Cas9编辑体系对T细胞PD-1基因编辑效率。方法:在前期研究基础上,通过优化电转缓冲液和脉冲条件组合,对CD8~+ T细胞的PD-1基因进行敲除编辑,检测电穿孔后CD8~+ T细胞凋亡、分化、增殖和PD-1表达情况。结果:与传统电穿孔方案(P3电转染缓冲液+EO115脉冲)相比,优化方案(P2电转染缓冲液+EH100脉冲)能显著提高PD-1敲除效率,但不影响T细胞存活、分化状态以及增殖。结论:P2电转染缓冲液+EH100脉冲的组合对T细胞PD-1基因的编辑效率较高。
Aim: To increase the gene editing efficiency of PD-1 on T cells by optimizing electroporation transfection conditions. Methods: Based on the previous research,by optimizing the combination of primary cell nucleofection solution and pulse conditions,we knocked out PD-1 gene from CD8~+ T cells,and detected CD8~+ T cell apoptosis,proliferation,differentiation,and PD-1 expression after electroporation. Results: Compared with traditional electroporation transfection protocol(P3 primary cell nucleofection solution + EO115 pulse),the optimized protocol(P2 primary cell nucleofection solution +EH100 pulse) did not affect T cell survival,proliferation,and differentiation,but significantly increased PD-1 knockout efficiency. Conclusion: The gene editing efficiency of PD-1 on T cells based on the CRISPR/Cas9 system can be improved by the optimized protocol of P2 primary cell nucleofection solution combined with EH100 pulse.
Aim: To increase the gene editing efficiency of PD-1 on T cells by optimizing electroporation transfection conditions. Methods: Based on the previous research,by optimizing the combination of primary cell nucleofection solution and pulse conditions,we knocked out PD-1 gene from CD8~+ T cells,and detected CD8~+ T cell apoptosis,proliferation,differentiation,and PD-1 expression after electroporation. Results: Compared with traditional electroporation transfection protocol(P3 primary cell nucleofection solution + EO115 pulse),the optimized protocol(P2 primary cell nucleofection solution +EH100 pulse) did not affect T cell survival,proliferation,and differentiation,but significantly increased PD-1 knockout efficiency. Conclusion: The gene editing efficiency of PD-1 on T cells based on the CRISPR/Cas9 system can be improved by the optimized protocol of P2 primary cell nucleofection solution combined with EH100 pulse.
引文
[1]MAIMELA NR,LIU SS,ZHANG Y.Fates of CD8+Tcells in tumor microenvironment[J].Comput Struct Biotechnol J,2019,17:1
[2]LUKE JJ,OTT PA.PD-1 pathway inhibitors:the next generation of immunotherapy for advanced melanoma[J].Oncotarget,2015,6(6):3479
[3]KEIR ME,BUTTE MJ,FREEMAN GJ,et al.PD-1 and its ligands in tolerance and immunity[J].Annu Rev Immunol,2008,26:677
[4]PUNKOSDY GA,BLAIN M,GLASS DD,et al.Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens[J].Proc Natl Acad Sci USA,2011,108(9):3677
[5]WANG WS,LAU R,YU DH,et al.PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+CD25(Hi)regulatory T cells[J].Int Immunol,2009,21(9):1065
[6]MALI P,ESVELT KM,CHURCH GM.Cas9 as a versatile tool for engineering biology[J].Nat Methods,2013,10(10):957
[7]GAJ T,GERSBACH CA,BARBAS CF.ZFN,TALEN,and CRISPR/Cas-based methods for genome engineering[J].Trends Biotechnol,2013,31(7):397
[8]LIU XJ,ZHANG YP,CHENG C,et al.CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells[J].Cell Res,2017,27(1):154
[9]SEKI A,RUTZ S.Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells[J].J Exp Med,2018,215(3):985
[10]任飞飞,李峰,李砺锋,等.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,2017,12(12):1362
[11]田永贵,张震,尹婕,等.不同亚群CD8+T细胞表型、功能分子及分化相关基因的表达[J].郑州大学学报(医学版),2019,54(3):318
[12]黄岚,张毅.肿瘤微环境中T细胞功能障碍及其逆转策略[J].中国肿瘤生物治疗杂志,2017,11(11):1247
[13]SANMAMED MF,CHEN LP.A Paradigm shift in cancer immunotherapy:from enhancement to normalization[J].Cell,2018,175(2):313
[14]MALI P,YANG LH,ESVELT KM,et al.RNA-Guided human genome engineering via Cas9[J].Science,2013,339(6121):823
[15]HENDEL A,BAK RO,CLARK JT,et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells[J].Nat Biotechnol,2015,33(9):985
[16]GOMES-SILVA D,SRINIVASAN M,SHARMA S,et al.CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-Cell malignancies[J].Blood,2017,130(3):285
[17]REN JT,LIU XJ,FANG CY,et al.Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition[J].Clin Cancer Res,2017,23(9):2255
[18]SCHUMANN K,LIN S,BOYER E,et al.Generation of knock-in primary human T cells using Cas9 ribonucleoproteins[J].Proc Natl Acad Sci USA,2015,112(33):10437
[2]LUKE JJ,OTT PA.PD-1 pathway inhibitors:the next generation of immunotherapy for advanced melanoma[J].Oncotarget,2015,6(6):3479
[3]KEIR ME,BUTTE MJ,FREEMAN GJ,et al.PD-1 and its ligands in tolerance and immunity[J].Annu Rev Immunol,2008,26:677
[4]PUNKOSDY GA,BLAIN M,GLASS DD,et al.Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens[J].Proc Natl Acad Sci USA,2011,108(9):3677
[5]WANG WS,LAU R,YU DH,et al.PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+CD25(Hi)regulatory T cells[J].Int Immunol,2009,21(9):1065
[6]MALI P,ESVELT KM,CHURCH GM.Cas9 as a versatile tool for engineering biology[J].Nat Methods,2013,10(10):957
[7]GAJ T,GERSBACH CA,BARBAS CF.ZFN,TALEN,and CRISPR/Cas-based methods for genome engineering[J].Trends Biotechnol,2013,31(7):397
[8]LIU XJ,ZHANG YP,CHENG C,et al.CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells[J].Cell Res,2017,27(1):154
[9]SEKI A,RUTZ S.Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells[J].J Exp Med,2018,215(3):985
[10]任飞飞,李峰,李砺锋,等.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,2017,12(12):1362
[11]田永贵,张震,尹婕,等.不同亚群CD8+T细胞表型、功能分子及分化相关基因的表达[J].郑州大学学报(医学版),2019,54(3):318
[12]黄岚,张毅.肿瘤微环境中T细胞功能障碍及其逆转策略[J].中国肿瘤生物治疗杂志,2017,11(11):1247
[13]SANMAMED MF,CHEN LP.A Paradigm shift in cancer immunotherapy:from enhancement to normalization[J].Cell,2018,175(2):313
[14]MALI P,YANG LH,ESVELT KM,et al.RNA-Guided human genome engineering via Cas9[J].Science,2013,339(6121):823
[15]HENDEL A,BAK RO,CLARK JT,et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells[J].Nat Biotechnol,2015,33(9):985
[16]GOMES-SILVA D,SRINIVASAN M,SHARMA S,et al.CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-Cell malignancies[J].Blood,2017,130(3):285
[17]REN JT,LIU XJ,FANG CY,et al.Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition[J].Clin Cancer Res,2017,23(9):2255
[18]SCHUMANN K,LIN S,BOYER E,et al.Generation of knock-in primary human T cells using Cas9 ribonucleoproteins[J].Proc Natl Acad Sci USA,2015,112(33):10437