Additional file 3 of Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair
Creators: name: Li, Guoling
name: Yang, Xiaohui
name: Luo, Xinxin
name: Wu, Zhenfang
name: Yang, Huaqiang
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 3: Figure S1. Reporter system monitoring HDR efficiency. A CRISPR/Cas9‐induced KI with dsDNA donor (circular or linear) with homology arms in GAPDH locus. LA, left arm, RA, right arm. B CRISPR/Cas9‐induced KI with ssODN donor to repair EGFP sequence. 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. 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. 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. 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 and error bars (SD) were calculated from three experiments. **P &lt; 0.01 compared to DMSO‐treated control group. 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. Raw data for qPCR test of mRNA expression shown in Fig. 5A. Data are mean ± SD from 3 or 4 technical replicates. **P &lt; 0.01 compared to DMSO‐treated control. Table S1. Oligoes and primers used in this study.