期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 114, 期 35, 页码 9409-9414出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1707635114
关键词
CRISPR/Cas9; Cas9-activators; gain-of-function; CRISPR mutagenesis; CRISPRa
资金
- NIH [R01GM084947, R24OD021997]
- NIH under Ruth L. Kirschstein National Research Service Award from the NIH General Medical Sciences Division [F32GM113395]
- Howard Hughes Medical Institute (HHMI) Exceptional Research Opportunities Program
- Brazil Scientific Mobility Program
- NIH National Center Institute Cancer Center Support Grant [5 P30 CA06516]
- National Key Technology Research and Development Program of the Ministry of Science and Technology of the People's Republic of China [2015BAI09B03, 2016YFE0113700]
- National Basic Research Program (973 Program) [2013CB35102]
- National Natural Science Foundation of China [31571320]
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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