title: Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans.
abstract: Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30-60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Alexandre
authors.identifier: http://orcid.org/0000-0002-8080-7546
authors.initials: A
authors.lastname: Paix
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Yuemeng
authors.identifier: http://orcid.org/0000-0003-3928-6926
authors.initials: Y
authors.lastname: Wang
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Harold E
authors.initials: HE
authors.lastname: Smith
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Chih-Yung S
authors.identifier: http://orcid.org/0000-0003-0982-2150
authors.initials: CY
authors.lastname: Lee
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Deepika
authors.identifier: http://orcid.org/0000-0002-0859-5390
authors.initials: D
authors.lastname: Calidas
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Tu
authors.identifier: http://orcid.org/0000-0002-5697-300X
authors.initials: T
authors.lastname: Lu
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Jarrett
authors.identifier: http://orcid.org/0000-0001-6354-6551
authors.initials: J
authors.lastname: Smith
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
authors.forename: Helen
authors.identifier: http://orcid.org/0000-0002-3449-2790
authors.initials: H
authors.lastname: Schmidt
authors.affiliation: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Krause
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205 gseydoux@jhmi.edu.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Seydoux
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism*; D018388:Codon, Terminator; D053903:DNA Breaks, Double-Stranded; D017353:Gene Deletion; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D016678:Genome; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D009841:Oligonucleotides; D059767:Recombinational DNA Repair
chemical_list: D064130:CRISPR-Associated Proteins; D018388:Codon, Terminator; D009841:Oligonucleotides
keywords: CRISPR; Caenorhabditis elegans; Cas9; genome editing; homology-directed repair; short homology arms