title: CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene.
abstract: The CRISPR/Cas technology has been successfully used to stimulate the integration of small DNA sequences in a target locus to produce gene mutations. However, many applications require homologous recombination using large gene-targeting constructs. Here we address the potential of CRISPR/Cas-mediated double-strand breaks to enhance the genetic engineering of large target sequences using a construct for "humanizing" the mouse Cnr2 gene locus. We designed a small-guide RNA that directs the induction of double strand breaks by Cas9 in the Cnr2 coding exon. By co-transfection of the CRISPR/Cas system with the 10 kb targeting construct we were able to boost the recombination frequency more than 200-fold from 0.27% to 67%. This simple technology can thus be used for the homologous integration of large gene fragments and should greatly enhance our ability to generate any kind of genetically altered mouse models.
authors.affiliation: Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gennequin
authors.forename: David-Marian
authors.initials: DM
authors.lastname: Otte
authors.forename: Andreas
authors.initials: A
authors.lastname: Zimmer
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D005091:Exons; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics; D043885:Receptor, Cannabinoid, CB2 / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection
chemical_list: C571431:Cnr2 protein, mouse; D043885:Receptor, Cannabinoid, CB2; D012313:RNA
keywords: Cannabinoid receptor; ES cells; Gene replacement; Knockout; Surveyor assay