Fig. 1

Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue