Fig. 3

Genome editing in iPSCs at the CTNNB1 locus with conventional vs. double cut HDR donors of 50–2000 bp in HA length. a Schematic of genome editing at the CTNNB1 locus. The double strand break (DSB) is created by Cas9/sgCTNNB1 39 bp before the stop codon TAA (marked in red). The donors contain a GS-mNeonGreen-Wpre-polyA sequence sandwiched by left HA (yellow shadow) and right HA (blue shadow). GS is a linker. Silent mutations (lowercase and bold) were introduced to prevent cleavage by Cas9/sgCTNNB1. sgCTNNB1 sequence: bold; cut site: green triangle; stop codon: red; backbone: lowercase. b FACS analysis of iPSCs three days after nucleofection. The percentages of mNeonGreen⁺ cells represent the HDR efficiencies. c Effects of HA length of conventional and double cut donors on HDR efficiency at the CTNNB1 locus. n = 4. d Schematic of different knockin patterns. Apart from being edited by HDR, linearized insert sequence or backbone sequence can also integrate into the locus through incomplete HDR (HDR at one side and NHEJ at the other side) or NHEJ. A pair of primers (red arrows) was used to amplify edited sequence. The amplicon size is shown at the right side. For NHEJ knockin patterns, the length of PCR product is imprecise because NHEJ might be accompanied by indels. e Procedure for knockin pattern analysis. PCR was carried out for 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 with different HA lengths. h Quantitative PCR (qPCR) analysis of donor plasmid backbone-forward insertion. y-axis indicates the relative ratio of NHEJ/HDR, in which NHEJ was calculated by qPCR data and HDR by the percentage of mNeonGreen⁺ cells in a certain sample. Primers (F2 and R2) for qPCR analysis are indicated in blue in (d). n = 3. c, h Error bars represent S.E.M. *P ≤ 0.05; **P ≤ 0.01; ns not significant, by Student’s paired t-test