Fig 2: SNV detection in HGPS cells.a Schematic of HGPS pathogenic point mutation. b Schematic of ABE editing of HGPS fibroblasts. c Representative Sanger sequencing results of untreated and ABE-treated HGPS fibroblasts. The black arrow indicates the HGPS pathogenic point mutation site. d and e, sgGOLDFISH in parallel with progerin immunofluorescence using (d) gLMNA-MUT or (e) gLMNA-WT. f, g Quantifications of progerin immunofluorescence intensity from different populations. Each dot represents a quantified cell. In Fig. 4f, n = 113, 69, 107 for Untreated, Mutant-positive, 1:1 mixture, respectively. In Fig. 4g, n = 152, 44, 50 for 1:1 mixture, Correction-positive, ABE treated, respectively. Mann-Whitney U Test (two-sided) is used. n.s. represents p > 0.05. The exact p values are 0.91225 (Untreated and Mutant-positive in Fig. 4f), 0.040809 (Mutant-positive and 1:1 mixture in Fig. 4f), 0.000008 (1:1 mixture and Correction-positive in Fig. 4g), 0.001048 (Correction-positive and ABE treated in Fig. 4g). Box represents the range of 25th to 75th percentiles, and whisker represents the range of 10th to 90th percentiles. Median line is shown in the box. A.U., arbitrary units. h Schematic of measuring distance from a FISH spot to the nuclear edge. i Quantifications of the relative distance (i.e., the distance from a FISH spot to the nuclear edge divided the square root of the nuclear area) of LMNA-WT, LMNA-MUT and MUC4 alleles to the nuclear edge. Each dot represents a quantified FISH spot (n = 538, 636 and 994 for LMNA-MUT, LMNA-WT and MUC4, respectively). Student’s t test (two-sided) is used. p = 1.83 × 10-17 (LMNA-MUT and MUC4) and 1.04*10-23 (LMNA-WT and MUC4), respectively. Median line is shown. Whisker represents mean ± SD. Raw data points underlying each plot and Sanger sequencing traces are provided as in Source Data files.

Fig 3: LMNA co-regulated genes prior to and after SID. (A) Interaction network of LMNA co-regulated genes during myogenesis (Supplementary Material, Table S2). The network was created via STRING, and proteins are clustered via MCL clustering (minimum required interaction score 0.400). Common genes with impaired expressions after SID in two patient iPSCs were marked with (*). (B) Heatmap of gene expression in iPSCs prior to and after SID for LMNA co-regulated genes. All gene expression was normalized to healthy control prior to SID. Gene expressions were clustered using correlation distance and average linkage (raw data in Table S3). Common genes with impaired expressions after SID in two patient iPSCs were marked within the black frame. (C) PCA analysis of gene expression file after SID of healthy and patient-derived iPSCs revealed three separated groups. (D,E) Selected gene markers among LMNA co-regulated genes for the next step gene editing (N = 3; p < 0.0001, ****). Two healthy controls (healthy1 and healthy2) and two clones from patient carrying N456D mutation (N456D_C1, N456D_C2) were included in validating the selected gene expression. Healthy1 and healthy2: hiPSCLMNA_WT/LMNA_WT; W498C: patient hiPSCsLMNAc.1494G>T/LMNA_WT; N456D_C1 or C2: patient hiPSCsLMNAc.1366A>G/LMNA_WT clone 1 or clone 2.

Fig 4: sgGOLDFISH.a Only one guide RNA (gMUC4-OneMM or gMUC4-TwoMM) and 23 FISH probes were used to target the MUC4-NR region. The figure shows the case that gMUC4-OneMM was used (there is one mismatch between guide RNA and target DNA). In contrast, although only one guide RNA (gMUC4-R) and one FISH probe species were used to target MUC4-R region, the MUC4-R region contains ~200 repeats, therefore multiple binding sites for eCas9 nickase RNP complexed with gMUC4-R and the FISH probe against MUC4-R region. b A representative sgGOLDFISH image using gMUC4-OneMM. Single cells outlined in orange are magnified on the upper–right corner, which show (i) two detected MUC4-NR alleles, (ii) one detected MUC4-NR allele (iii.) no detected MUC4-NR allele. Histograms of sgGOLDFISH MUC4-NR foci per cell using gMUC4-OneMM (n = 78) or gMUC4-TwoMM (n = 100). c Quantification of co-localized MUC4-R and MUC4-NR foci. d Representative sgGOLDFISH images using gACTB-OneMM and gACTB-TwoMM, and histograms of sgGOLDFISH ACTB foci using gACTB-OneMM (n = 195) or gACTB-TwoMM (n = 203). e Representative sgGOLDFISH images using gLMNA-WT and gLMNA-MUT, and histograms of sgGOLDFISH LMNA foci using gLMNA-WT (n = 121) or gLMNA-MUT (n = 110). Raw data points underlying each plot are provided as a Source Data file.

Fig 5: Impaired expression of LMNA after serum-induced differentiation in patient-derived iPSCs. (A) Scheme of SID protocol. (B) Unstained and stained iPSC LMNA N456D_C1 cells prior to and after SID, prior to SID as SID(-) and after SID as SID(+). Cells were stained with anti-lamin A/C antibody (green). Scale bar for unstained images 400 µm, for stained images 10 µm. (C–E) Western blot of lamin A/C protein expression in healthy and patient iPSCs prior to and after SID (N = 3; p < 0.0001, ****). A second healthy control iPSC and a second clone for patient iPSC LMNA N456D were also included (Figure S2D) for quantification, and the level of healthy1 after SID was used as reference, with grey bars as prior to SID and blue bars are SID. (F–H) LMNA mRNA expression in healthy and patient iPSCs prior to and after SID (N = 3; p < 0.0001, **** except N456D_C2). Sequencing results of reverse transcription (RT) products from mRNAs of healthy and patient iPSC prior to SID. The sequencing results after the SID can be seen in Figure S3. For quantification, the level of healthy1 after SID was used as reference with grey bars prior to SID, while blue bars are SID. Healthy1 and healthy2: two healthy controls hiPSCLMNA_WT/LMNA_WT; W498C: patient hiPSCsLMNAc.1494G>T/LMNA_WT; N456D_C1 or C2: patient hiPSCsLMNAc.1366A>G/LMNA_WT clone 1 or clone 2.