title: Creating Heritable Mutations in Drosophila with CRISPR-Cas9.
abstract: Reverse genetics-the creation of mutations in preselected target genes-has until recently been a bottleneck in many Drosophila projects. The advent of clustered, regularly interspaced, short palindromic repeat (CRISPR) genome engineering systems has transformed this situation. A short time after the in vitro demonstration of target site cleavage by the RNA-guided endonuclease CRISPR-associated nuclease 9 (Cas9) (Jinek et al., Science 337:816-821, 2012), hundreds of fly researchers are using CRISPR technology to generate loss-of-function mutant alleles in specific genes, as well as to create specific point mutations or tagged protein products. It appears that most target genes can be edited with remarkably high efficiency, with engineered strains often available a few weeks after conception of a project. Here, we provide a step-by-step protocol for creating loss-of-function mutations in Drosophila using transgenic Cas9 sources, which is based on optimized reagents and procedures that have been evaluated in our laboratory. We also provide guidance on extending this protocol to produce precise genomic alterations by homology-directed repair in the presence of a donor sequence. Additional information and updates are available from our website, www.crisprflydesign.org .
authors.affiliation: Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.|Division of Signaling andFunctional Genomics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.
authors.forename: Fillip
authors.initials: F
authors.lastname: Port
authors.affiliation: Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK. f.port@dkfz.de.
authors.forename: Simon L
authors.initials: SL
authors.lastname: Bullock
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing; D017344:Genes, Insect; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D016678:Genome; D054643:INDEL Mutation; D016297:Mutagenesis, Site-Directed; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D059767:Recombinational DNA Repair; D059386:Reverse Genetics / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D004262:DNA Restriction Enzymes
keywords: CRISPR-Cas9; Drosophila; Genome engineering; Genotyping; Indel; gRNA design and cloning