title: High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.
abstract: Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.
authors.affiliation: Howard Hughes Medical Institute (HHMI) and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 apaix1@jhmi.edu.
authors.forename: Alexandre
authors.identifier: http://orcid.org/0000-0002-8080-7546
authors.initials: A
authors.lastname: Paix
authors.affiliation: Howard Hughes Medical Institute (HHMI) and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Andrew
authors.initials: A
authors.lastname: Folkmann
authors.affiliation: Howard Hughes Medical Institute (HHMI) and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Dominique
authors.identifier: http://orcid.org/0000-0003-2210-1569
authors.initials: D
authors.lastname: Rasoloson
authors.affiliation: Howard Hughes Medical Institute (HHMI) and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Seydoux
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D017173:Caenorhabditis elegans / Q000235:genetics*; D004260:DNA Repair; D005818:Genetic Engineering / Q000379:methods*; D007267:Injections; D030781:Organisms, Genetically Modified; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012261:Ribonucleoproteins / Q000008:administration & dosage*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012261:Ribonucleoproteins
keywords: C. elegans; CRISPR-Cas9; genome editing; homology-directed repair; ribonucleoprotein complexes