title: Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes.
abstract: Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4-5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments.
authors.affiliation: FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Norris
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Hyun-Min
authors.initials: HM
authors.lastname: Kim
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 mcolaiacovo@genetics.med.harvard.edu jcalarco@fas.harvard.edu.
authors.forename: Mónica P
authors.initials: MP
authors.lastname: Colaiácovo
authors.affiliation: FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138 mcolaiacovo@genetics.med.harvard.edu jcalarco@fas.harvard.edu.
authors.forename: John A
authors.initials: JA
authors.lastname: Calarco
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D017173:Caenorhabditis elegans / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D018390:Gene Targeting; D016678:Genome; D005838:Genotype; D059765:Homologous Recombination / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*; D059767:Recombinational DNA Repair / Q000235:genetics
keywords: C. elegans; CRISPR; Cas9; genome editing