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, 194 (4), 1029-35

Genome Engineering of Drosophila With the CRISPR RNA-guided Cas9 Nuclease

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Genome Engineering of Drosophila With the CRISPR RNA-guided Cas9 Nuclease

Scott J Gratz et al. Genetics.

Abstract

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.

Keywords: CRISPR RNA; Cas9; Drosophila; genome engineering; homologous recombination.

Figures

Figure 1
Figure 1
Cas9-generated DSBs are repaired by NHEJ and HR in Drosophila embryos. (A) Schematic of the yellow locus indicating YE1, Y5′, and Y3′ chiRNA target sites. The predicted cut site for each chiRNA is indicated (red arrows). Inset shows schematic of CRISPR RNA/Cas9 system components interacting with target DNA. For additional detail, see Figure S1A. (B) Results of the SURVEYOR assay indicate that targeted cleavage occurred in yellow at the predicted YE1 cut site (left, embryos injected with YE1 chiRNA; right, control, C, embryos). Duplexes lacking indels were uncut (open arrowhead), whereas duplexes containing indels were cut asymmetrically at the targeted site (solid arrowheads). One of 13 embryos injected with YE1 chiRNA produced duplexes with indels. (C) Schematic of the rosy locus indicating R1 and R2 chiRNA target sites (red arrows). (D) Results from SURVEYOR assays indicate R1 and R2 chiRNAs generated indels at the targeted cut sites (embryos injected with R1 chiRNA, R2 chiRNA, or control, C, are as indicated). Uncut duplexes (open arrowhead) and cut duplexes (solid arrowhead) are indicated. Three of four embryos injected with either R1 chiRNA or R2 chiRNA produced duplexes with indels. (E) The yellow locus was amplified from embryos injected with Cas9, Y5′ chiRNA, and Y3′ chiRNA using the primers indicated in A (open arrows). Targeted deletions result in a 650-bp PCR product (C, uninjected control embryo). (F) Products from nine PCRs were pooled for sequencing. Sequence alignments reveal breakpoints and imprecise repair between predicted cut sites (red arrowheads). One repair event that resulted in the 4.6-kb deletion of yellow plus an indel deleting an additional 27 bp and inserting 93 bp is not shown. (G) Schematic of the yellow ssODN design. The ssODN includes 60-nt homology arms (red) flanking a 50-nt attP docking site (purple). The homology arms correspond to the sequences immediately adjacent to the predicted cut sites (red arrows). For additional detail see Figure S1C. (H) The yellow locus was amplified from embryos injected with Cas9, Y5′ chiRNA, Y3′ chiRNA, and ssODN using the primers indicated in A. HR events in which the locus is deleted and replaced with attP will yield a 700-bp product. Products from three reactions were pooled for sequence analysis, which confirmed the replacement of yellow with attP in five of eight clones.
Figure 2
Figure 2
CRISPR-induced modifications are efficiently transmitted through the germline. (A) Representative sequences of independent yellow mutants generated by Y1E-guided Cas9-induced DSBs. The predicted YE1 cleavage site is indicated with a red arrowhead. Short sequence repeats flanking the cleavage site (underlined in the wild-type sequence) may have facilitated the preferential generation of the same 4-bp deletion in three independent founders by MMEJ. (B) The yellow locus was amplified from yellow progeny of males and females injected with Cas9, Y5′ chiRNA, and Y3′ chiRNA using the primers indicated in Figure 1A. Fifteen independent progeny are represented (C, no template control). A 650-bp PCR product is expected (solid arrowhead) if a targeted deletion of yellow has been transmitted. PCR products corresponding to partial deletions (shaded arrowheads), and wild-type (open arrowhead) yellow are indicated. (C) The yellow locus was amplified from yellow progeny of flies injected with Cas9, Y5′ chiRNA, Y3′ chiRNA, and ssODN using the primers indicated in Figure 1A. Seven independent progeny are represented (C, no template control). A 700-bp product is expected following the targeted replacement of yellow with attP. PCR products corresponding to correct HR events are indicated (solid arrowheads) and were confirmed by Sanger sequencing.

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