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.
['Zhang|Fan|F|0000-0002-7186-6317', 'Cheng|De||0000-0003-3712-6232', 'Wang|Shuwen|S|', 'Zhu|Jiyue|J|0000-0003-2171-0725']
D000818:Animals D064113:CRISPR-Cas Systems D053903:DNA Breaks, Double-Stranded D000072669:Gene Editing D059765:Homologous Recombination D006801:Humans D051379:Mice D010957:Plasmids
Crispr/Cas9 mESC TERT knock-in