CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene

• Published in: Biochemical and biophysical research communications 2013-11, Vol.441 (4), p.815-819
• Main Authors: Gennequin, Benjamin, Otte, David-Marian, Zimmer, Andreas
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Cannabinoid receptor; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; ES cells; Exons; Gene replacement; Genetic Engineering - methods; Genetic Loci; Humans; Knockout; Mice; Molecular Sequence Data; Receptor, Cannabinoid, CB2 - genetics; Recombination, Genetic - genetics; RNA - genetics; Surveyor assay; Transfection
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.10.138

•A CRISPR/Cas system was designed to induce a double-strand breaks in the mouse Cnr2 locus.•This system increased the frequency of homologous recombination more than 200 fold.•The high recombination frequency resulted in 67% correctly targeted ES cell clones with a “humanized” CB2 receptor gene.•Four out of 63 targeted clones exhibited bi-allelic recombination. 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 10kb 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.