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Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76. doi: 10.1073/pnas.1405500111. Epub 2014 Jul 7.

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

Author information

1
Division of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; and fport@mrc-lmb.cam.ac.uk sbullock@mrc-lmb.cam.ac.uk.
2
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147.

Abstract

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

PMID:
25002478
PMCID:
PMC4115528
DOI:
10.1073/pnas.1405500111
[Indexed for MEDLINE]
Free PMC Article

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