Biotechnology and bioengineeringMay 26, 2020

CRISPR cuts help precise DNA repair when editing large chromosome regions

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 13, 2026

Abstract

Homologous recombination efficiency reached up to 16% in mouse embryonic stem cells using Crispr/Cas9 technology.

An 18.1-kb genomic region around the mTert gene was replaced with a 45.5-kb recombinant fragment.
A total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles were obtained.
The Crispr/Cas9 system induced DNA double-strand breaks, leading to increased homologous recombination efficiency.
High rates of genomic DNA deletions and mutations were observed at the target sites due to Crispr/Cas9 cleavages.
This approach may provide a feasible method for manipulating large chromosomal regions in mammalian cells.

Simplified

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.

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CONFLICT OF INTEREST. None of the authors have professional or financial affiliations that could be perceived to bias the presentation of this manuscript.

PubMed

CRISPR cuts help precise DNA repair when editing large chromosome regions

Abstract

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Abstract

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