Optimized strategy for in vivo Cas9-activation in Drosophila


Ewen-Campen B, Yang-Zhou D, Fernandes VR, González DP, Liu L-P, Tao R, et al. Optimized strategy for in vivo Cas9-activation in Drosophila. Proc Natl Acad Sci U S A. 2017;114 (35) :9409-9414.

Date Published:

2017 Aug 29


While 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.

Last updated on 09/21/2017