PNAS：美杜克大学研究学者利用CRISPR技术表达限制HIV传染限制因子

摘要 : 12月14日，国际著名期刊《美国国家科学院院刊》在线发表杜克大学的Bryan R. Culle教授发表的一篇研究论文，研究人员用CRISPR/Cas9技术构建了转录激活子，成功让人类细胞表达了自己缺乏的限制因子(A3G和A3B)。 研究人员发现，诱导A3G或A3B高水平表达需要使用两个sgRNA。这两种限制因子都能阻断Vif缺陷型HIV-1的复制，但只有A3B能抑制野生型HIV-1传染性。因为 A3G会被HIV-1的Vif蛋白降解，而A3B能够抵抗Vif。 研究表明，基于CRISPR的转录激活子可以有效激活调控病毒复制的人类基因。人们可以用它们在基因组中筛选能够影响病毒复制的内源基因。如果找到有效的递送途径，这一技术还有望用于临床治疗。 原文链接： Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators 原文摘要： Whereas several mammalian proteins can restrict the re

12月14日，国际著名期刊《美国国家科学院院刊》在线发表杜克大学的Bryan R. Culle教授发表的一篇研究论文，研究人员用CRISPR/Cas9技术构建了转录激活子，成功让人类细胞表达了自己缺乏的限制因子(A3G和A3B)。

研究人员发现，诱导A3G或A3B高水平表达需要使用两个sgRNA。这两种限制因子都能阻断Vif缺陷型HIV-1的复制，但只有A3B能抑制野生型HIV-1传染性。因为 A3G会被HIV-1的Vif蛋白降解，而A3B能够抵抗Vif。

研究表明，基于CRISPR的转录激活子可以有效激活调控病毒复制的人类基因。人们可以用它们在基因组中筛选能够影响病毒复制的内源基因。如果找到有效的递送途径，这一技术还有望用于临床治疗。

原文链接：

Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators

原文摘要：

Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such “synergistic activation mediators” can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functionalvif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed.

doi: 10.1073/PNAS.1516305112

作者：Bryan R. Cullen

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