Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organism...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 47; pp. 19012 - 19017
Main Authors:	Ren, Xingjie, Sun, Jin, Housden, Benjamin E., Hu, Yanhui, Roesel, Charles, Lin, Shuailiang, Liu, Lu-Ping, Yang, Zhihao, Mao, Decai, Sun, Lingzhu, Wu, Qujie, Jie, Jun-Yuan, Xi, Jianzhong, Mohr, Stephanie E., Xu, Jiang, Perrimon, Norbert, Ni, Jian-Quan
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 19-11-2013 NATIONAL ACADEMY OF SCIENCES National Acad Sciences
Subjects:	Animals Animals, Genetically Modified Biological Sciences Comparative analysis CRISPR-Cas Systems - genetics Databases, Genetic Deoxyribonucleic acid DNA Drosophila Drosophila melanogaster Drosophila melanogaster - genetics Drosophila Proteins - genetics Embryos Genetic engineering Genetic Engineering - methods Genetic mutation Genomes Genomics Genomics - methods Germ Cells Insects Mutagenesis Mutagenesis - genetics Plasmids Promoter Regions, Genetic - genetics Ribonucleic acid RNA RNA-Binding Proteins - genetics nanos-Cas9 HRMA
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Summary:	The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.
Bibliography:	http://dx.doi.org/10.1073/pnas.1318481110 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Contributed by Norbert Perrimon, October 8, 2013 (sent for review September 5, 2013) 1X.R. and J.S. contributed equally to this work. Author contributions: J. Xu, N.P., and J.-Q.N. designed research; X.R., J.S., B.E.H., L.-P.L., Z.Y., D.M., L.S., Q.W., J. Xu, N.P., and J.-Q.N. performed research; Y.H., C.R., S.L., J.-Y.J., and J. Xi contributed new reagents/analytic tools; X.R., J.S., B.E.H., L.-P.L., Z.Y., D.M., L.S., Q.W., J. Xu, and J.-Q.N. analyzed data; and S.E.M., J. Xu, N.P., and J.-Q.N. wrote the paper.
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.1318481110