3 years ago

Optimized strategy for in vivo Cas9-activation in Drosophila [Genetics]

Optimized strategy for in vivo Cas9-activation in Drosophila [Genetics]
Norbert Perrimon, Ben Ewen–Campen, Vitoria R. Fernandes, Jian–Quan Ni, Jonathan Zirin, Yanhui Hu, Stephanie E. Mohr, Jin Sun, Delfina P. Gonzalez, Donghui Yang–Zhou, Xingȷie Ren, Rong Tao, Lu–Ping Liu

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.

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