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. 2015;1311:335-48.
doi: 10.1007/978-1-4939-2687-9_22.

Precise Genome Editing of Drosophila With CRISPR RNA-Guided Cas9

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Free PMC article

Precise Genome Editing of Drosophila With CRISPR RNA-Guided Cas9

Scott J Gratz et al. Methods Mol Biol. .
Free PMC article

Abstract

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.

Figures

Fig. 1
Fig. 1
Experimental overview. Injection mixtures containing pHsp70-Cas9, the appropriate pBS-U6-gRNA(s), and, for gene replacement, an ssDNA donor are injected into pre-blastoderm embryos. Twenty-four hours after injection, a subset of embryos can be assayed molecularly for the presence of targeted modifications to assess efficiency. Rear remaining embryos to adulthood and outcross to recover heritable modifications. Methods for identifying flies with the targeted modification include (1) phenotypic screening if the phenotype of the targeted modifications is known and readily observable, (2) molecular screening by PCR-based analysis, (3) negative screening for the removal of a marked transposable elements in the targeted locus, and (4) positive screening using a dsDNA donor with a visible marker
Fig. 2
Fig. 2
gRNA cloning. The pBS-U6-gRNA vector contains two recognitions sites for the type IIs restriction enzyme BbsI. Following BbsI digestions, unique 4-nt overhangs mediate seamless cloning targeting sequences into the gRNA backbone. Targeting sequence inserts are generated as two 5′ phosphorylated oligonucleotides that are annealed to create complementary overhangs (5′-CTTC, top strand; 5′-AAAC, bottom strand) for ligation into linearized pBS-U6-gRNA
Fig. 3
Fig. 3
ssDNA design. Single-stranded oligonucleotide donors are designed to contain approximately 60 nt of homology to the target locus sequences immediately adjacent to each predicted Cas9-mediated DSB. The homology regions flank a 50-nt attP sequence that provides subsequent access to the targeted locus. Red arrowheads mark the predicted Cas9 cleavage sites. Target site sequences (green) and PAMs (blue) are indicated

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