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Fly (Austin). 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.

Author information

1
Department of Biochemistry and Molecular Biology; Mayo Clinic; Rochester, MN USA; Division of Molecular Biology and Biochemistry; University of Missouri-Kansas City; Kansas City, MO USA.
2
Graduate Program in Neurobiology of Disease; Mayo Graduate School; Mayo Clinic; Rochester, MN USA.
3
Department of Biochemistry and Molecular Biology; Mayo Clinic; Rochester, MN USA.
4
Department of Biochemistry and Molecular Biology; Mayo Clinic; Rochester, MN USA; Division of Gastroenterology and Hepatology; Mayo Clinic; Rochester, MN USA.

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

PMID:
24141137
PMCID:
PMC3974895
DOI:
10.4161/fly.26828
[Indexed for MEDLINE]
Free PMC Article

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