Fig. 6: Effect of Cas9TX on genome stability.

a Immunofluorescence for γH2AX in HEK293T cells treated with etoposide, SpCas9, or Cas9TX. Images were taken 24 h after transfection by confocal microscopy. Both SpCas9 and Cas9TX were expressed with a P2A-mCherry tag. mCherry alone was the negative control. b Statistics for γH2AX foci in each HEK293T cell treated with etoposide, SpCas9, or Cas9TX. N = 100 cells were analyzed for each group, two-tailed t-test, ****p < 0.00001; n.s, no significance. c Indel counts for Cas9-edited and Cas9TX-edited mESCs by WGS analysis. Three biological replicates, two-tailed t-test. n.s means no significance. Values from minimum to maximum are shown in the box. The line represents the average. d The impacts of SpCas9 and Cas9TX on the editing efficiency and translocation levels of AsCas12a:C-MYC3 detected by PEM-seq in HEK293T cells. Mean ± SD from three biological replicates. Two-tailed t-test, **p < 0.01. e Editing efficiency for SpCas9 and Cas9TX at C-MYC2 and the identified C-MYC2 off-target site detected by PEM-seq in HEK293T cells. Mean ± SD from three biological replicates. Two-tailed t-test, **p < 0.01. DNA sequences for C-MYC2 and the C-MYC2 off-target are shown at the top. Mismatched DNA is in red. f Percentages of translocations cloned from the C-MYC2 off-target site by PEM-seq in HEK293T cells. Mean ± SD from three biological replicates. Two-tailed t-test, **p < 0.01. g Editing frequency of SpCas9 and Cas9TX at the on-targets and off-targets of VEGFA and EMX1 identified by PEM-seq analysis in Figs. 3 and 4. Primers were designed at off-target sites with PCR amplification. Editing efficiency was evaluated by TIDE. Fold changes for Cas9TX to SpCas9 are at the top. Mean ± SD from three replicates. Source data are provided as a Source Data file.