Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing.
Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing.
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DOI：
10.4049/jimmunol.1701616
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发表时间：
2018-09-01
期刊：
影响因子：
--
通讯作者：
Unutmaz D
中科院分区：
文献类型：
--
作者：
Chen X;Kozhaya L;Tastan C;Placek L;Dogan M;Horne M;Abblett R;Karhan E;Vaeth M;Feske S;Unutmaz D
在原代人类T细胞亚群中使用CRISPR开发精确有效的基因编辑方法将为解码其功能提供有效的工具。为了实现这一目标，我们利用慢病毒CRISPR/Cas9系统使原代人T细胞稳定表达Cas9基因和靶向感兴趣基因的编码或非编码区的指导RNA（gRNA）。我们发现，多个基因（CD 4，CD 45，CD 95）可以同时和稳定地删除在幼稚，记忆，效应或调节（Treg）T细胞subets在非常高的效率。此外，与转录激活因子或阻遏物相关的核酸酶缺陷型Cas9（dCas 9）可以下调或增加T细胞中基因的表达。例如，糖蛋白A重复占优势（GARP）（一种通常且排他地在活化的T细胞上表达的基因）的表达可以通过融合至转录激活因子的dCas 9在非Treg效应T细胞上诱导。进一步的分析表明，这种方法可以用于定位基因转录中涉及的启动子序列。通过这种CRISPR/Cas9介导的基因编辑，我们还在几个实例中证明了人T细胞功能分析的可行性：（1）CD 95缺失在再活化时抑制T细胞凋亡;（2）ORAI 1（一种Ca 2+释放激活通道）的缺失，消除了Ca 2+内流和细胞因子分泌，模拟了免疫缺陷患者中的天然基因突变;（3）CD 25或CD 127表达的转录激活分别增强了IL-2或IL-7的细胞因子信号传导。总之，将CRISPR工具箱应用于人类T细胞亚群对于解码其功能输出的机制具有重要意义。
Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Towards this goal, we utilized lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs (gRNA) that targeted either coding or non-coding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naïve, memory, effector or regulatory (Treg) T cell susbets at very high efficiency. In addition, nuclease-deficient Cas9 (dCas9), associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of Glycoprotein A repetitions predominant (GARP), a gene which is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by dCas9 fused to transcriptional activators. Further analysis determined that this approach could be utilized in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9 mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: (1) CD95 deletion inhibited T cell apoptosis upon reactivation; (2) deletion of ORAI1, a Ca2+ release-activated channel, abolished Ca2+ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; (3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Together, the application of CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional ouputs.
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影响因子：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
影响因子：
5.2
作者：
Feske S
通讯作者：
Feske S
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
64.8
作者：
Prakriya, Murali;Feske, Stefan;Hogan, Patrick G.
通讯作者：
Hogan, Patrick G.
DOI：
10.1073/pnas.1013285108
发表时间：
2011-02-22
影响因子：
11.1
作者：
Maul-Pavicic, Andrea;Chiang, Samuel C. C.;Ehl, Stephan
通讯作者：
Ehl, Stephan