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Fig. 6 | Genome Biology

Fig. 6

From: Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage

Fig. 6

Regulating cell cycle further improves HDR efficiency of the double cut donor system. a The effects of small molecules on HDR efficiency at the CTNNB1 or PRDM14 locus. The iPSCs were treated with RS-1 (10 μM), Nu7441 (2 μM), SCR7 (1 μM), Brefeldin A (0.1 μM), L755507 (5 μM), or Nocodazole (100 ng/mL) at 0–24 h after nucleofection and the HDR efficiency was determined by FACS on day 3. b The effects of RAD51, Ad4E1B-Eorf46, and CCND1 on HDR efficiency at the CTNNB1 or PRDM14 locus. The plasmid encoding RAD51, Ad4E1B-Eorf46, or CCND1 was co-transfected with Cas9, sgRNA, and pDonor. The HDR efficiency was examined by FACS on day 3. c The effects of Nocodazole and CCND1 on HDR efficiency at the CTNNB1 or PRDM14 locus. The plasmid encoding CCND1 was co-transfected with Cas9, sgRNA, and donor plasmid. Nocodazole (100 ng/mL) was added into the medium at 0–24 h after transfection. HDR efficiency was determined by FACS on day 3. ac Comparison with control by Student’s paired t-test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns not significant. df CCND1 increases HDR rate at the CTNNB1 locus. The procedure for knockin pattern analysis was detailed above. At least 30 colonies were picked for Sanger sequencing at both ends

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