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. 2014;8(1):52-7.
doi: 10.4161/fly.26828. Epub 2013 Oct 18.

A Simplified and Efficient Germline-Specific CRISPR/Cas9 System for Drosophila Genomic Engineering

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Free PMC article

A Simplified and Efficient Germline-Specific CRISPR/Cas9 System for Drosophila Genomic Engineering

Zachary L Sebo et al. Fly (Austin). .
Free PMC article

Abstract

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.

Keywords: CRISPR/Cas9; RNA-guided DNA cleavage; engineered endonuclease; genomic engineering; germline.

Figures

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Figure 1. Disrupting EGFP and mRFP genes using a germline-specific CRISPR/Cas9 system. (A) Scheme for generating Vasa-Cas9 transgenic flies and designing EGFP and mRFP gRNAs. The germline-specific Cas9 expressing vector, pVasa-Cas9, was integrated into the ZH-2A site on chromosome X mediated by the phiC31 integrase/attP/attB integration system. The resulting Vasa-Cas9 strain contains the germline-specific Vasa-driven Cas9 sequence and the eye promoter 3xP3-driven EGFP and mRFP sequences. Two gRNAs were designed to target mRFP and EGFP, respectively. Blue letters indicate the gRNA spacer sequences. The 3′ common regions of the gRNAs are represented with identical hairpin structures. Bold magenta labels the PAM motifs of the DNA substrates. Cyan triangles indicate the Cas9 cut sites. Key chromogenic residues are labeled red and green respectively. (B) EGFP and mRFP expression in G1 adult eyes. Representative images of G1 adult eyes from Vasa-Cas9 G0 flies injected with EGFP (top panels) or mRFP (bottom panels) gRNA-expressing vectors. Six flies are shown in each condition: those that retain fluorescent signals in top rows, and those showing a loss of fluorescent signals in bottom rows. EGFP signals are shown on the left, mRFP in the middle, and their overlays with bright-field signals on the right. (C) Scoring of EGFP and mRFP mutation germline transmission rates. EGFP (left panel) and mRFP (right panel) gRNA-induced mutation germline transmission rates are sorted from lowest (100% wild type) to highest (100% mutant). Each bar represents G1 scoring of an individual cross from a single injected G0 Vasa-Cas9 fly. Twenty-one G0 crosses (total 465 G1 flies) were scored for EGFP and 48 G0 crosses (total 1418 G1 flies) were scored for mRFP gRNA-induced G1 mutations. Yellow portions indicate the percentage of G1 flies expressing both EGFP and mRFP (wild type), red portions indicate mRFP only expression (EGFP mutant), and green portions indicate EGFP only expression (mRFP mutant). Fifteen out of 21 (71%) G0 EGFP-gRNA vector injected adults and 17 out of 48 (35%) G0 mRFP-gRNA vector injected adults harbor transmittable G1 germline mutations. Two EGFP- and 1 mRFP-gRNA vector injected G0adults produced 100 percent G1 mutant progenies.

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