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Fly (Austin). 2013 Oct-Dec;7(4):249-55. doi: 10.4161/fly.26566. Epub 2013 Oct 2.

CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand.

Author information

1
Genetics Training Program; University of Wisconsin-Madison; Madison, WI USA.
2
Department of Biochemistry; University of Wisconsin-Madison; Madison, WI USA.
3
Department of Biomolecular Chemistry; University of Wisconsin School of Medicine and Public Health; Madison, WI USA.
4
Genetics Training Program; University of Wisconsin-Madison; Madison, WI USA; Laboratory of Genetics; University of Wisconsin-Madison; Madison, WI USA; Laboratory of Cell and Molecular Biology; University of Wisconsin-Madison; Madison, WI USA.

Abstract

The CRISPR/Cas9 system has attracted significant attention for its potential to transform genome engineering. We and others have recently shown that the RNA-guided Cas9 nuclease can be employed to engineer the Drosophila genome, and that these modifications are efficiently transmitted through the germline. A single targeting RNA can guide Cas9 to a specific genomic sequence where it induces double-strand breaks that, when imperfectly repaired, yield mutations. We have also demonstrated that 2 targeting RNAs can be used to generate large defined deletions and that Cas9 can catalyze gene replacement by homologous recombination. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have shown similar promise in Drosophila. However, the ease of producing targeting RNAs over the generation of unique sequence-directed nucleases to guide site-specific modifications makes the CRISPR/Cas9 system an appealingly accessible method for genome editing. From the initial planning stages, engineered flies can be obtained within a month. Here we highlight the variety of genome modifications facilitated by the CRISPR/Cas9 system along with key considerations for starting your own CRISPR genome engineering project.

KEYWORDS:

CRISPR; Cas9; genome engineering; homologous recombination; site-directed mutagenesis

PMID:
24088745
PMCID:
PMC3896497
DOI:
10.4161/fly.26566
[Indexed for MEDLINE]
Free PMC Article

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