title: Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair.
abstract: As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8-9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35-80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158 jordan.ward@ucsf.edu.
authors.forename: Jordan D
authors.identifier: http://orcid.org/0000-0001-9870-8936
authors.initials: JD
authors.lastname: Ward
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D005785:Gene Conversion; D017353:Gene Deletion; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods; D017238:Genes, Helminth; D005804:Genes, Lethal; D005818:Genetic Engineering / Q000379:methods*; D049751:Genome, Helminth; D008297:Male; D009154:Mutation; D017393:RNA Editing; D034622:RNA Interference; D018510:RNA, Helminth; D059767:Recombinational DNA Repair
chemical_list: D018510:RNA, Helminth
keywords: CRISPR/Cas9; co-conversion; nonhomologous end joining; oligonucleotide-mediated homologous recombination; pha-1