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 cell-biology disciplines.

功能获得性研究通常需要将转基因cDNA繁琐地克隆到载体中，以进行超出生理表达水平的过表达。CRISPR / Cas技术的快速发展为解决这些问题提供了广阔的机遇。在这里，我们报告了一种简单的无克隆方法，可使用CRISPR / Cas9激活剂在内源性基因座上诱导基因表达。我们的策略是利用合成的sgRNA表达盒来指导与转录激活因子（VP64，p65和Rta）融合的核酸酶无效的Cas9复合物，以特异性地诱导内源基因。这种策略允许在同一天快速启动功能获得研究。使用这种方法，我们测试了两个CRISPR激活系统dSpCas9VPR和dSaCas9VPR，以诱导人和大鼠细胞中的多个基因。人细胞中的CRX，RORB，RAX，OTX2，ASCL1和NEUROD1），而在三种不同的基因（Ascl1，Neurod1，Nrl）中观察到，大鼠细胞的基因诱导水平更高，而效率更低。总之，这项研究提供了一种使用CRISPR / Cas9激活剂有效激活内源基因表达的简单方法，该方法可以用作快速工作流程来启动一系列分子生物学和细胞生物学学科的功能获得研究。