Mao Decai, Jia Yu, Peng Ping, Shen Da, Ren Xingjie, Zhu Ruibao, Qiu Yuhao, Han Yuting, Yu Jinchao, Che Qinyun, Li Yutong, Lu Xinyi, Liu Lu-Ping, Wang Zhao, Liu Qingfei, Sun Jin, Ni Jian-Quan
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
Sichuan Academy of Grassland Science, Chengdu 611731, China.
G3 (Bethesda). 2020 Dec 3;10(12):4483-4488. doi: 10.1534/g3.120.401614.
The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.
flySAM/CRISPRa系统最近已成为在功能获得性研究中一种强大的工具。该系统包括Gal4/UAS驱动的dCas9激活剂和U6启动子控制的sgRNA。在确定dCas9激活剂优于其他组合后，为进一步提高靶向激活剂的效率，我们系统地优化了sgRNA的参数。有趣的是，发现最有效的sgRNA在转录起始位点（TSS）上游-150bp至-450bp区域积累，并且激活效率与sgRNA靶向序列的GC含量呈强正相关。此外，靶区域对GC含量起主导作用，因为靶向距TSS超过-600bp区域的sgRNA即使含有75%的GC也会失去效率。令人惊讶的是，当比较靶向两条DNA链的sgRNA的活性时，无论是在细胞中还是在[此处原文缺失信息]，靶向非模板链的sgRNA都优于与模板链互补的sgRNA。总之，我们定义了sgRNA设计标准，这将极大地促进CRISPRa在功能获得性研究中的应用。
