Ewen-Campen, BenjaminYang-Zhou, DonghuiFernandes, Vitória R.González, Delfina P.Liu, Lu-PingTao, RongRen, XingjieSun, JinHu, YanhuiZirin, JonathanMohr, StephanieNi, Jian-QuanPerrimon, Norbert2021-09-292017-08-29Ewen-Campen, Benjamin, Donghui Yang-Zhou, Vitória R. Fernandes, Delfina P. González, Lu-Ping Liu, Rong Tao, Xingjie Ren et al. "Optimized Strategy for in Vivo Cas9-Activation in Drosophila." Proceedings of the National Academy of Sciences 114, no. 35 (2017): 9409-9414. DOI: 10.1073/pnas.17076351140027-84241091-6490https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37369614While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.en-USResearch Subject Categories::NATURAL SCIENCES::Biology::Other biology::Functional genomicsJournal Article2021-09-2910.1073/pnas.1707635114