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Contributed by Norbert Perrimon, October 8, 2013 (sent for review September 5, 2013)
Significance
Using the recently introduced Cas9/sgRNA technique, we have developed a method for specifically targeting Drosophila germ-line cells to generate heritable mutant alleles. We have established transgenic lines that stably express Cas9 in the germ line and compared different promoters and scaffolds of sgRNA in terms of their efficiency of mutagenesis. An overall mutagenesis rate of 74.2% was achieved with this optimized system, as determined by the number of mutant progeny out of all progeny screened. We also evaluated the off-targets associated with the method and established a Web-based resource, as well as a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Our results demonstrate that this optimized Cas9/sgRNA system in Drosophila is efficient, specific, and cost-effective and can be readily applied in a semi-high-throughput manner.
Abstract
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.
Footnotes
↵1X.R. and J.S. contributed equally to this work.
- ↵2To whom correspondence may be addressed. E-mail: jiangxu{at}mail.tsinghua.edu.cn, perrimon{at}receptor.med.harvard.edu, or nijq{at}mail.tsinghua.edu.cn.
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.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1318481110/-/DCSupplemental.
Optimized Cas9 gene editing for Drosophila
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