title: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.
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.
authors.affiliation: 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.
authors.forename: Fillip
authors.initials: F
authors.lastname: Port
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Chen
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147.
authors.forename: Tzumin
authors.initials: T
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Simon L
authors.initials: SL
authors.lastname: Bullock
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004260:DNA Repair; D004331:Drosophila melanogaster / Q000235:genetics*; D018390:Gene Targeting; D020043:Genes, Essential; D017344:Genes, Insect / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D049750:Genome, Insect / Q000235:genetics*; D005854:Germ Cells / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012313:RNA / Q000235:genetics; D019076:Transgenes / Q000235:genetics
chemical_list: D012313:RNA