A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9
A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9
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DOI：
10.1016/j.omtn.2018.11.008
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发表时间：
2019-03-01
影响因子：
8.8
通讯作者：
Wong, Raymond C. B.
中科院分区：
文献类型：
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作者：
Fang, Lyujie;Hung, Sandy S. C.;Wong, Raymond C. B.
功能获得研究通常需要将转基因cDNA克隆到载体中，以便在生理表达水平之外进行过表达。CRISPR/Cas技术的快速发展为解决这些问题提供了有希望的机会。在这里，我们报道了一种使用CRISPR/Cas9激活剂在内源性位点诱导基因表达的简单、无克隆的方法。我们的策略是利用合成的sgRNA表达盒来引导一个无核酸酶的Cas9复合体与转录激活因子（VP64、p65和Rta）融合，以实现内源基因的位点特异性诱导。这一策略允许在同一天内快速启动功能获得研究。利用这种方法，我们测试了两种CRISPR激活系统，dSpCas9VPR和dSaCas9VPR，用于在人和大鼠细胞中诱导多种基因。我们的研究结果表明，这两种CRISPR激活剂都可以在人类细胞中有效诱导六种不同的神经发育基因（CRX、RORB、RAX、OTX2、ASCL1和NEUROD1），而在大鼠细胞中，正如在三种不同的基因（ASCL1、NEUROD1、Nrl）中观察到的那样，基因诱导水平更加可变，效率更低。总之，本研究提供了一种使用CRISPR/Cas9激活剂有效激活内源性基因表达的简单方法，可作为一种快速工作流程，用于启动一系列分子和细胞生物学学科的功能获得性研究。
Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular-and cellbiology disciplines.