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Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair. | LitMetric

Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair.

Cell Biosci

National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China.

Published: November 2023

AI Article Synopsis

  • Gene knock-in (KI) via homology-directed repair (HDR) is challenging due to inefficiency in animal cells, particularly since HDR is more effective during specific cell cycle phases (S and G2/M).
  • Researchers tested various cell cycle inhibitors (docetaxel, irinotecan, nocodazole, and mitomycin C) to synchronize cells in these phases, finding that they enhanced CRISPR/Cas9-mediated KI in both animal cells and embryos.
  • The study discovered that these inhibitors activate common pathways linking the cell cycle to DNA repair, ultimately boosting factors necessary for HDR and suggesting potential for improved genetically modified animals and gene therapy strategies.

Article Abstract

Background: Gene knock-in (KI) in animal cells via homology-directed repair (HDR) is an inefficient process, requiring a laborious work for screening from few modified cells. HDR tends to occur in the S and G2/M phases of cell cycle; therefore, strategies that enhance the proportion of cells in these specific phases could improve HDR efficiency.

Results: We used various types of cell cycle inhibitors to synchronize the cell cycle in S and G2/M phases in order to investigate their effect on regulating CRISPR/Cas9-mediated HDR. Our results indicated that the four small molecules-docetaxel, irinotecan, nocodazole and mitomycin C-promoted CRISPR/Cas9-mediated KI with different homologous donor types in various animal cells. Moreover, the small molecule inhibitors enhanced KI in animal embryos. Molecular analysis identified common signal pathways activated during crosstalk between cell cycle and DNA repair. Synchronization of the cell cycle in the S and G2/M phases results in CDK1/CCNB1 protein accumulation, which can initiate the HDR process by activating HDR factors to facilitate effective end resection of CRISPR-cleaved double-strand breaks. We have demonstrated that augmenting protein levels of factors associated with the cell cycle via overexpression can facilitate KI in animal cells, consistent with the effect of small molecules.

Conclusion: Small molecules that induce cell cycle synchronization in S and G2/M phases promote CRISPR/Cas9-mediated HDR efficiency in animal cells and embryos. Our research reveals the common molecular mechanisms that bridge cell cycle progression and HDR activity, which will inform further work to use HDR as an effective tool for preparing genetically modified animals or for gene therapy.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10676593PMC
http://dx.doi.org/10.1186/s13578-023-01159-4DOI Listing

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