Optimized strategy for in vivo Cas9-activation in Drosophila .

Autor:	Ewen-Campen B; Department of Genetics, Harvard Medical School, Boston, MA 02115., Yang-Zhou D; Department of Genetics, Harvard Medical School, Boston, MA 02115., Fernandes VR; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Federal University of Sao Joao del Rei, Divinopolis, Minas Gerais 36301, Brazil., González DP; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Pomona College, Claremont, CA 91711., Liu LP; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Drosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115.; Howard Hughes Medical Institute, Boston, MA 02115., Tao R; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Drosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115., Ren X; Gene Regulatory Laboratory, School of Medicine, Tsinghua University, Beijing 100084, China., Sun J; Gene Regulatory Laboratory, School of Medicine, Tsinghua University, Beijing 100084, China., Hu Y; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Drosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115., Zirin J; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Drosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115., Mohr SE; Department of Genetics, Harvard Medical School, Boston, MA 02115.; Drosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115., Ni JQ; Gene Regulatory Laboratory, School of Medicine, Tsinghua University, Beijing 100084, China nijq@mail.tsinghua.edu.cn perrimon@receptor.med.harvard.edu., Perrimon N; Department of Genetics, Harvard Medical School, Boston, MA 02115; nijq@mail.tsinghua.edu.cn perrimon@receptor.med.harvard.edu.; Howard Hughes Medical Institute, Boston, MA 02115.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2017 Aug 29; Vol. 114 (35), pp. 9409-9414. Date of Electronic Publication: 2017 Aug 14.
DOI:	10.1073/pnas.1707635114
Abstrakt:	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. Competing Interests: The authors declare no conflict of interest.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu