Fig. 5

High HDR efficiency at the PRDM14 locus is achieved by double cut HDR donor with short HA. a Schematic of genome editing at the PRDM14 locus. An sgPRDM14 was designed to create DSB at 4 bp after the stop codon TAG (marked in red). The donors contain a 2A-GFP-Wpre-polyA sequence sandwiched by left HA (marked in yellow shadow) and right HA in blue shadow. The PRDM14 and GFP open reading frames (ORF) are fused by a ribosome skipping sequence 2A. pD-GFP is a conventional circular HDR donor and pD-GFP-sg is a double cut HDR donor flanked with the sgPRDM14 target sequence. sgPRDM14 sequence: bold; cut site: red triangle; plasmid backbone: lowercase. b Determination of HDR by FACS. iPSCs were analyzed three days after nucleofection of Cas9, sgPRDM14 together with either pD-GFP or pD-GFP-sg. The percentages of GFP⁺ cells represent the HDR efficiencies. c Effects of HA length of conventional and double cut donors on HDR efficiency at the PRDM14 locus. n = 4; error bars represent S.E.M. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns not significant, by Student’s paired t-test. d Schematic of different knockin patterns. Apart from being edited by HDR, cells could also be integrated with linearized inert sequence or backbone sequence through incomplete HDR or NHEJ. Two pairs of primers were designed to amplify the edited locus. The amplicon size is listed at the right side. e Procedure for knockin pattern analysis. PCR was carried out twice. The bands between the 2000–4000 bp area were cut off and cloned into pJET vector and individual bacterial colonies were picked for Sanger sequencing. f Summary of Sanger sequencing results. g Distribution of different knockin patterns by double cut HDR donors. h Determination of monoallelic vs. biallelic HDR by donor pD-GFP-sg-HA300-300 bp. Twelve edited clones were analyzed