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

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. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-023-01159-4.


Additional file 3
Captions for supplementary figures and table Figure S1 ssODN is chemically modified with PS linkage at both ends to optimize the DNA repair efficiency.The EGFP-repaired ssODN sequence is shown in Table S1.Table S1.Oligoes and primers used in this study.

Figure S2 .
Figure S2.Test of CRISPR/Cas9-mediated HDR efficiency in BHK-21 and PFF cells with DOC, NOC, IRI and MITO treatment.Circular dsDNA donor, linear dsDNA donor and ssODN donor were separately used in BHK-21 (A) and PFF cells (B).The cells were transfected with CRISPR and reporter for 12 h and then treated with small molecules for 48 h.HDR efficiency is demonstrated by EGFP positivity tested by flow cytometry.Control are cells with reporter transfection and then DMSO treatment for the same time.Data are mean ± SD from 2 or 3 independent experiments.

Figure S3 .
Figure S3.Test of the combinational use the four small molecules on HDR efficiency.The four small molecules in different combinations were used to treat 293T (A), BHK-21 (B) and PFF (C) transfected with CRISPR/Cas9 and circular dsDNA, linear dsDNA or ssODN donor.HDR efficiency is shown by the percentage of EGFP-positive cells.Control are cells with reporter transfection and then DMSO treatment for the same time.Data are mean ± SD.Each dot represents an independent experiment.

Figure S4 .
Figure S4.Small molecule effects on ssODN-mediated KI in Apoe and Sox2 loci of BHK-21 cells.A. The donor is a 146 nt ssODN that is homologous to the target sequence and contains a 6 nt insertion (HindIII restriction sequence) at the CRISPR cleavage site.B. The KI frequency after 48 h-treatment with different small molecules was determined by HindIII digestion of PCR products covering the KI site.The ratio of cleaved products to total DNA substrate (cleaved PCR bands + uncleaved PCR band) is KI frequency.A T7E1 digestion of the same PCR product was used an inner control to show all targeting events including HDR and NHEJ.C. Quantification of KI frequency of cells with different small molecule treatments by estimating band density shown in B by Image J software.The mean values

Figure S5 .
Figure S5.Immunofluorescence assay of protein tagging frequency with small molecule treatment.The strategy inserting 6 × His tag into N terminals of Sod1 (A) and Ku70 (B) genes in BHK-21 cell.After 12 h-transfection and then small molecule treatment for 48 h, cells were immunostained with anti-His antibody to show the abundance of tagged proteins.Enhanced fluorescence signals [red for His-SOD1 (A) and green for His-KU70 (B)] can be found in small molecule-treated cells compared to DMSO-treated cells, demonstrating enhanced tagging frequency in the two loci by small molecule treatment.Scale bars: 50 μm.

Figure S6 .
Figure S6.Raw data for qPCR test of mRNA expression shown in Figure 5A.Data are mean ± SD from 3 or 4 technical replicates.**P < 0.01 compared to DMSO-treated control.

Table S1 .
Oligoes and primers used in this study