Crispr/Cas9‐mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1‐kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double‐strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single‐guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination‐mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions. Extremely low efficiency is a bottleneck for the manipulation of large chromosomal regions in genome editing by homologous recombination. In this study, the authors designed a strategy that allowed the modification of large segments of mouse genome by combining the traditional homologous recombination approach with Crispr/Cas9‐assisted genome cleavages. The data demonstrated the feasibility and efficiency of this strategy by successfully replacing an 18‐kb genomic region at the mTert locus with a 45‐kb chimeric hTERT/mTert fragment in mouse embryonic stem cells.