Fig. 3

Small molecules significantly promote CRISPR-Cpf1-mediated generation of knockin hPSC lines without drug selection. a A scheme of the targeting strategy of OCT4-2A-tdTomato. b FACS analysis for OCT4-tdTomato-positive cells after transfection of the OCT4-tdTomato donor plasmid and the OCT4-targeting crRNA into hPSCs. A representative FACS result of samples treated with DMSO, VE-822, and AZD-7762 was shown. c Addition of VE-822 and AZD-7762 increased the percentage of OCT4-tdTomato-positive cells. n = 3 experiments. Statistical significance calculated using two-tailed Student’s t-test, compared to DMSO controls. *P < 0.05, **P < 0.01 ***P < 0.001. d Characterization of OCT4-tdTomato reporter lines. Scale bar, 100 μM. e FACS analysis of OCT4-tdTomato reporter lines. Undifferentiated line is in red, and differentiated line is in purple. f PCR genotyping of OCT4-tdTomato reporter lines. The size of expected bands is 1131 bp. g Sequencing results of the non-targeted allele and at the junction of correctly targeted allele in OCT4-tdTomato reporter lines. h A scheme of ssODN-mediated genome editing. We used ALKBH1–cr1 and a synthesized 120-nt ssODN template to introduce point mutations, which could be detected by RFLP assay. i The RFLP assay result. NcoI was used to evaluate the ssODN-mediated knockin efficiency. j VE-822 and AZD-7762 could promote ssODN-mediated genome editing by almost 3-fold. n = 4 experiments. Statistical significance calculated using two-tailed Student’s t-test, compared to DMSO controls. ****P < 0.0001. k Sanger sequencing confirmed the successful introduction of point mutations