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Crispr/Cas9‐mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region
Author(s) -
Zhang Fan,
Cheng De,
Wang Shuwen,
Zhu Jiyue
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27441
Subject(s) - crispr , homologous recombination , cas9 , genome editing , biology , genetics , gene targeting , gene , homologous chromosome , non homologous end joining , dna , genomic dna , recombination , microbiology and biotechnology
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.