Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region
- 16 June 2020
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 117 (9), 2816-2826
- https://doi.org/10.1002/bit.27441
Abstract
Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanizedmTertallele,hmTert, in the mouse genome by replacing an 18.1-kb genomic region around themTertgene 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 heterozygoushmTert/mTertalleles and three clones with homozygoushmTertalleles. 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.Funding Information
- National Institutes of Health (R01GM071725)
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