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.
Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

PMID:

 

24211574

 

[PubMed – as supplied by publisher]