ArticleBiotechnology and bioengineering2020
Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region.
Article in Biotechnology and bioengineering, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 15 citations in OpenAlex.
- Taxonomic and functional diversity of the microbiome associated with the freshwater sponge Metania sp. (Haplosclerida: Metaniidae) from the Brazilian Cerrado, a metagenomic approach.BMC microbiology · 2026Article
- CRISPR-Cas targeting inmicroLife · 2026Article
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- Genetic Engineering of Humanized Telomere Mice.Bio-protocol · 2025Article
- Modification of the telomerase gene with human regulatory sequences resets mouse telomeres to human length.Nature communications · 2025Article
- Humanization of the mouseResearch square · 2024Article
- Deep mutational scanning of the RNase III-like domain inFrontiers in cellular and infection microbiology · 2024Article
- Therapeutic Potential of CRISPR/Cas in Hashimoto's Thyroiditis: A Comprehensive Review.Current gene therapy · 2024Review
- An easy method to generate recombinant pseudorabies virus expressing the capsid protein of Porcine circovirus type 2d.Frontiers in microbiology · 2023Article
- Genome Editing Targets for Improving Nutrient Use Efficiency and Nutrient Stress Adaptation.Frontiers in genetics · 2022Review
- Review
- CRISPR-Cas technology based genome editing for modification of salinity stress tolerance responses in rice (Oryza sativa L.).Molecular biology reports · 2021Review
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
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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