Journal Article

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

Luo, Xinxin, Li, Guoling, Yang, Xiaohui, Wu, Zhenfang, Yang, Huaqiang

2023

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Animal behavior / Animal genetic engineering / Biomedical and Life Sciences / CCNB1 / CDK1 / Cell Biology / Cell cycle / Cells / CRISPR / CRISPR/Cas9 / DNA / DNA repair / Drug dosages / Efficiency / Embryonic development / Embryos / Flow cytometry / Gene editing / Gene expression / Gene loci / Gene therapy / Genes / Genetic engineering / Genetically modified animals / Genomes / Homology / Homology-directed repair / Irinotecan / Life Sciences / Microbiology / Mitomycin / Mitomycin C / Molecular modelling / Negotiation, mediation and arbitration / Neurobiology / Nocodazole / Proteins / Proteomics / Scientific equipment and supplies industry / Stem Cells / Synchronization / Toxicity