title: CRISPR/Cas9 Genome Editing in Caenorhabditis elegans: Evaluation of Templates for Homology-Mediated Repair and Knock-Ins by Homology-Independent DNA Repair.
abstract: Precise genome editing by the Cas9 nuclease depends on exogenously provided templates for homologous recombination. Here, we compare oligonucleotides with short homology and circular DNA molecules with extensive homology to genomic targets as templates for homology-based repair of CRISPR/Cas9 induced double-strand breaks. We find oligonucleotides to be templates of choice for introducing small sequence changes into the genome based on editing efficiency and ease of use. We show that polarity of oligonucleotide templates greatly affects repair efficiency: oligonucleotides in the sense orientation with respect to the target gene are better templates. In addition, combining a gene loss-of-function phenotype screen with detection of integrated fluorescent markers, we demonstrate that targeted knock-ins in Caenorhabditis elegans also can be achieved by homology-independent repair.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland iskra.katic@fmi.ch.
authors.forename: Iskra
authors.initials: I
authors.lastname: Katic
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.
authors.forename: Lan
authors.initials: L
authors.lastname: Xu
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.
authors.forename: Rafal
authors.initials: R
authors.lastname: Ciosk
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D017173:Caenorhabditis elegans / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics; D055879:Gene Knock-In Techniques; D049751:Genome, Helminth; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010641:Phenotype
chemical_list: D001426:Bacterial Proteins; D004270:DNA, Circular; D009841:Oligonucleotides; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: C. elegans; CRISPR; Cas9; NHEJ; genome editing