293T and BHK-21 (ATCC) were grown in DMEM (high glucose, Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific) and GlutaMAX (Thermo Fisher Scientific) at 37 °C with 5% CO2. Primary PFFs were isolated from a 30-day pig fetus, and grown in DMEM (high glucose) supplemented with 15% Fetal Bovine Serum and GlutaMAX at 39 °C with 5% CO2.

Three types of homologous templates were constructed separately to perform circular dsDNA, linear dsDNA and ssODN-mediated KI by coupling with corresponding CRISPR/Cas9 vector (based on eSpCas9(1.1), Addgene plasmid #71814) in cell transfection. For dsDNA homologous templates, the plasmids containing EGFP flanked by approximately 800-bp homology arms on both left and right sides of GAPDH target were used to insert EGFP to C terminal of GAPDH for an in-frame expression. The ssODN-mediated KI system contains a mutated EGFP expression vector disrrupted by an inserted gRNA sequence and a stop codon in the middle of the EGFP gene, and an ssODN with phosphorthioate (PS) modification in both ends to restore an intact EGFP sequence [28, 29]. The following ssODNs were used for KI manipulation in endogenous genes: 146 nt PS-modified ssODNs for HindIII KI in AAVS1 and SOD1 loci in 293T cells, Apoe and Sox2 loci in BHK-21 cells and ROSA26 locus in pig embryos; 158 nt PS-modified ssODNs for 6 × His tagging in N terminal of SOD1 and KU70 in 293T and BHK-21 cells. CDS sequences of human CCNA2, CCNB1, CDK1 and pig CDK1 were amplified by RT-PCR from cDNA of corresponding cells and cloned into pcDNA3.1(+) vector (Invitrogen) for overexpression. The ssODN, CRISPR-gRNA and related primer sequences were listed in Additional file 3: Table S1.

The dose-dependent effect and additive effect of small molecules were tested in multiple immortal cell lines including 293T and BHK-21 and primary cells from pig embryos. Cells were subcultured in suitable wells one day prior to transfection and transfected with donor and CRISPR/Cas9 vectors targeting corresponding loci with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s manual. Small molecules (Nocodazole, HY-13520; Docetaxel, HY-B0011; Irinotecan, HY-16562; and Mitomycin C, HY-13316. All from MCE) were added into cell culture at 12 h after transfection and cells were further incubated for 2 days to assay KI efficiency. The KI efficiency was represented by EGFP-positive rate determined by flow cytometry. The small molecule concentrations used in cells are 10 µM for irinotecan, 5 µM for docetaxel, 5 µM for mitomycin C, 2.5 µM for nocodazole and their mix except for the dose gradients indicated in Fig. 2 and Additional file 3: Fig. S2.

Cells were transfected with ssODN and CRISPR shown in “Vectors and reporter system” section and treated with small molecules for 48 h to identify KI efficiency. For HindIII KI in AAVS1 and SOD1 loci in 293T cells, as well as Apoe and Sox2 loci in BHK-21 cells, the primers were designed to amplify DNAs covering the HindIII restriction site introduced into the modified alleles (HindIII KI site PCR primers, Additional file 3: Table S1). KI efficiency was identified by HindIII digestion of PCR products, using T7E1 digestion as a control showing all editing events. For T7E1 assay, PCR product were hybridized in NEB Buffer 2 (New England Biolabs) using the following conditions: 95 °C for 5 min, 95–85 °C at − 2 °C/s, 85–25 °C at − 0.1 °C/s and held at 4 °C. Then, the hybridized PCR products were cut by T7E1 enzyme (New England Biolabs) and gel analyzed to determine the percent of total editing rate in the target alleles. The KI rate was calculated with HindIII digestion rate/T7E1 digestion rate. For detecting tagging efficiency of 6 × His at N terminal of SOD1 and KU70 in 293T and BHK-21 cells, cells were lysed for western blot or fixed for immunofluorescence using anti-His antibody after small molecule addition to the transfected cells for 48 h.

Pig embryo collection, culture and injection process were conducted as previously described [30]. In brief, pig ovaries were collected from a local slaughter house and cumulus oocyte complexes (COCs) were aspirated from antral follicles. After 3 times washing in maturation medium, COCs were cultured in a 4-well multi-dish (Nunc) at 39 °C in maturation medium in an atmosphere of 5% CO2 in air for 42 to 44 h. After the maturation culture, oocytes were freed from cumulus cells by vigorous vortexing. Cumulus-free oocytes were activated by an electric pulse, followed by 4 h of incubation in embryo culture medium (PZM3) containing 2 mM 6-Dimethylaminopurine (Sigma). The activated parthenogenetic embryos were subjected to cytoplasmic microinjection at 1-cell stage with the mixture containing spCas9 nuclease protein (New England Biolabs), synthesized Rosa26 sgRNA and ssODN donor at 300, 50 and 25 ng/μl, respectively. Approximately 10 picoliters of mixture was injected into each embryo. Injected embryos were washed three times in PZM3 and transferred to a 4-well multi-dish containing the same medium with small molecules and incubated at 39 °C in 5% CO2 in air for 6 days to reach blastocyst stage. Embryo was individually examined by PCR amplification of target site and HindIII digestion to determine modification status. All reagents for embryo manipulation are from Sigma unless otherwise stated.

The four small molecule-treated 293T cells and DMSO-treated control cells were subjected to total RNA extraction and RNAseq. In brief, the polyA mRNA was isolated for next generation sequencing library preparation. The libraries were loaded on Illumina NovaSeq 6000 and sequencing was carried out using a 2 × 150 bp paired-end configuration. The sequencing data were processed and aligned to Homo Sapiens reference genome (GRCh37) for further analysis of gene expression level, PCA, differentially expressed genes and functional enrichment. The sequencing was processed and analyzed by Azenta Life Sciences.

Total RNA was harvested from cell with TRIzol (Thermo Fisher Scientific) and reverse transcribed with PrimeScript RT reagent Kit with gDNA Eraser (Takara). qPCR was performed using TB Green Premix Ex Taq (Takara) in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) with the gene-specific primers (Additional file 3: Table S1). The relative mRNA expression of the tested genes was calculated using the 2(−ΔΔCt) method by normalizing to the ACTB mRNA level.

Cell samples were homogenized with RIPA buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (Sigma). Cell lysate supernatants were quantified with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and adjusted to an identical concentration using H2O. Equal amount of samples was boiled in SDS-PAGE loading buffer and loaded for SDS-PAGE. After protein separation in gel, proteins were transferred on a PVDF membrane (Millipore) under wet condition. The membrane was blocked in 5% non-fat milk in TBST buffer and then incubated with primary antibody labeling specific protein. After washing three times with TBST, the membrane was further incubated with HRP conjugated secondary antibody specific to the IgG of the species of primary antibody host. The target proteins were visualized with ECL (Thermo Fisher Scientific). The primary antibodies used in this study are as follows: anti-His-Tag (66005-1-Ig, Proteintech), anti-CCNA2 (18202-1-AP, Proteintech), anti-CCNB1 (55004-1-AP, Proteintech), anti-CDK1 (19532-1-AP, Proteintech), anti-Phospho-CDK1 (Tyr15) (#4539, Cell Signaling), anti-RAD51 (14961–1-AP, Proteintech), anti-CTIP (12624-1-AP, Proteintech), anti-RPA2 (10412-1-AP, Proteintech) and anti-β-actin (sc-47778, Santa Cruz Biotechnology).

BHK-21 cells were transfected with ssODN containing 6 × His tag and CRISPR/Cas9, and treated with the small molecules for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min followed by washing three times in PBS for 5 min each time. Then, cells were blocked in immunostaining blocking solution for 60 min followed by incubating cells with His-tag antibody at appropriate dilution in PBS overnight at 4 °C. After rinsing three times in PBS for 5 min each time, cells were further stained with fluorescence-conjugated secondary antibody at appropriate dilution in PBS for 1 h at 37 °C in darkness. Finally, the cells were thoroughly washed with PBS and examined by fluorescence microscopy.

Cell cycle distributions were determined by flow cytometry. 293T cells with small molecule treatments were trypsinized to single cells, washed in PBS and fixed in 70% ethanol at 4 °C for 30 min. The cells were then washed twice in PBS and incubated in a solution containing 20 μg/ml propidium iodide (PI, Sigma), 100 μg/ml RNase A (Sigma) and 0.05% Triton X-100 (Sigma) for 40 min in darkness at room temperature. Cell fluorescence was measured using an Accuri C6 Flow Cytometer (BD Biosciences). The cell areas representing G0/G1, S and G2/M phases in a DNA histogram were labeled and calculated.

All data are presented as mean values ± SD unless otherwise indicated. The means and SD are calculated from values of independent experiments, which are labeled as circles or dots on each bar in the plots unless otherwise indicated in figure legends. Statistical significance was determined by Student’s t test analysis (two-tailed) for two groups and one-way ANOVA with Dunnet’s post-hoc test for three or more groups. Differences in means were considered statistically significant at P < 0.05. Significance levels are: * P < 0.05; ** P < 0.01.