title: A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.
abstract: Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Heesun
authors.initials: H
authors.lastname: Kim
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Takao
authors.initials: T
authors.lastname: Ishidate
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Krishna S
authors.initials: KS
authors.lastname: Ghanta
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Meetu
authors.initials: M
authors.lastname: Seth
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Darryl
authors.initials: D
authors.lastname: Conte
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 masaki.shirayama@umassmed.edu craig.mello@umassmed.edu.
authors.forename: Masaki
authors.initials: M
authors.lastname: Shirayama
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605 masaki.shirayama@umassmed.edu craig.mello@umassmed.edu.
authors.forename: Craig C
authors.initials: CC
authors.lastname: Mello
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics*; D005819:Genetic Markers; D049751:Genome, Helminth; D059765:Homologous Recombination / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D064130:CRISPR-Associated Proteins; D029742:Caenorhabditis elegans Proteins; D005819:Genetic Markers; D003851:Deoxyribonucleases
keywords: CRISPR-Cas9 system; CRISPR-Cas9-induced indels; CRISPR-Cas9-mediated HR; Co-CRISPR; blasticidin selection