title: Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs.
abstract: Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.
authors.affiliation: Dept of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA apaix1@jhmi.edu.
authors.forename: Alexandre
authors.identifier: 0000-0002-8080-7546
authors.initials: A
authors.lastname: Paix
authors.affiliation: Dept of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: Schmidt
authors.affiliation: Dept of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA gseydoux@jhmi.edu.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Seydoux
mesh_terms: D000483:Alleles; D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D017173:Caenorhabditis elegans / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004274:DNA, Recombinant / Q000235:genetics*; D005785:Gene Conversion / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D059765:Homologous Recombination / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D059767:Recombinational DNA Repair / Q000235:genetics; D013698:Templates, Genetic
chemical_list: D064130:CRISPR-Associated Proteins; D004274:DNA, Recombinant; D009841:Oligonucleotides