Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9
The readily programmable CRISPR-Cas9 system is transforming genome engineering. We and others have adapted the S. pyogenes CRISPR-Cas9 system to precisely engineer the Drosophila genome and demonstrated that these modifications are efficiently transmitted through the germline. Here we provide a detailed protocol for engineering small indels, defined deletions, and targeted insertion of exogenous DNA sequences within one month using a rapid DNA injection-based approach.
Key wordsCRISPR Cas9 Genome engineering Nonhomologous end joining Homology-directed repair Drosophila
Clustered regularly interspaced short palindromic repeats
Nonhomologous end joining
Protospacer adjacent motif
Trans-activating CRISPR RNA
We are grateful to members of the Harrison, O’Connor-Giles, and Wildonger labs for their help in establishing CRISPR-Cas9 protocols in Drosophila, and to Dustin Rubinstein for comments on this chapter. Our work has been funded by start-up funds from the University of Wisconsin to M.M.H., J.W., and K.O.C.G. and grants from the National Institutes of Health to J.W. (R00 NS072252) and K.O.C.G. (R00 NS060985 and R01 NS078179). Plasmids and transgenic fly lines described here are available through the nonprofit distributor Addgene and the Bloomington Drosophila Stock Center, respectively. Detailed reagent information is available at http://flycrispr.molbio.wisc.edu.
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