@article {45063,
	title = {Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.},
	journal = {Proceedings of the National Academy of Sciences of the United States of America},
	volume = {111},
	year = {2014},
	month = {2014 Jul 22},
	pages = {E2967-76},
	abstract = {<p>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.</p>
},
	issn = {1091-6490},
	doi = {10.1073/pnas.1405500111},
	author = {Port, Fillip and Chen, Hui-Min and Lee, Tzumin and Bullock, Simon L}
}
