Ewen-Campen, B., Yang-Zhou, D., Fernandes, V.R., González, D.P., Liu, L.P., Tao, R., Ren, X., Sun, J., Hu, Y., Zirin, J., Mohr, S.E., Ni, J.Q., Perrimon, N. (2017). Optimized strategy for in vivo Cas9-activation in Drosophila.  Proc. Natl. Acad. Sci. U.S.A. 114(35): 9409--9414.

FBrf0236540

Research paper

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.

PMC5584449 (PMC) (EuropePMC)