Fig. 3

Small molecule effects on ssODN-mediated KI in endogenous genes of 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 for a simple identification of positive KI alleles by HindIII digestion. B The KI frequency after 48 h-treatment with different small molecules was determined directly 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, and a T7E1 digestion of the same PCR product was used an inner control to show all mutant alleles (NHEJ + HDR). C Quantification of KI frequency (up, AAVS1 locus; down, SOD1 locus) in 293T 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 < 0.01 compared to control group. D The strategy for gene tagging by CRISPR-induced ssODN-mediated KI. A 158 nt ssODN donor for tagging a 6 × His epitope in the N terminal of two genes (SOD1 and KU70) in 293T and BHK-21 cells. E The ssODN-mediated protein tagging effects were determined by western blot. Representative results showing increased 6 × His tagged proteins in small molecule-treated groups compared to DMSO-treated control