Optimized Strategy for in Vivo Cas9-Activation in Drosophila
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Fernandes, Vitória R.
González, Delfina P.
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CitationEwen-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.1707635114
AbstractWhile 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.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37369614
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