title: CRISPR-Cas9 Genome Editing in Drosophila.
abstract: The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.
authors.affiliation: Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Gratz
authors.affiliation: Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: C Dustin
authors.initials: CD
authors.lastname: Rubinstein
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Harrison
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Jill
authors.initials: J
authors.lastname: Wildonger
authors.affiliation: Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin.|Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin.|Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin.|Corresponding author: Kate M. O'Connor-Giles (oconnorgiles@wisc.edu).
authors.forename: Kate M
authors.initials: KM
authors.lastname: O'Connor-Giles
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004330:Drosophila / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D011995:Recombination, Genetic
keywords: CRISPR; Cas9; Drosophila; genome engineering; homology directed repair