Jia Yu, Shen Da, Wang Xia, Sun Jin, Peng Ping, Xu Rong-Gang, Xu Bowen, Ni Jian-Quan
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
Tsingdao Advanced Research Institute, Tongji University, Qingdao 266000, China.
Bio Protoc. 2019 Jan 20;9(2):e3147. doi: 10.21769/BioProtoc.3147.
Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in based on the structure-guided engineering of CRISPR-Cas9 complex.
能够增强基因表达的强大且通用的方法对于系统研究基因功能很有用。迄今为止，与生成功能丧失型突变体的方法相比，实现功能获得的方法有限。整个领域严重依赖于二十多年前开发的Gal4/UAS:cDNA过表达系统。克隆基因的编码DNA序列（CDS），尤其是那些大尺寸的基因，既费力又昂贵。此外，由于异位表达，经常会观察到这种方法的副作用。而且，用传统方法同时激活两个基因通常很耗时，对于三个或更多基因则很少能实现。在本方案中，我们描述了如何基于CRISPR-Cas9复合物的结构导向工程构建一个有效且便捷的靶向激活系统flySAM，以在果蝇中激活内源基因。
