Fig. 5: Investigating performance of GS-ABE8e using a plasmid-based reporter system.

a Targeting ABE8e and GS-ABE8e to a representative EGFP locus in HEK293T cells with gRNAs containing guides in various lengths. Data are presented as mean ±s.d. (n = 3). b EGFP disruption with guide sequences containing consecutive transversion mismatches. Data are presented as mean ±s.d. (n = 3). c Investigating base editors’ activities on out-of-protospacer A-to-G editing. 10 ng of EGFP(Q81stop) plasmid was used for each well. Data are presented as mean ±s.d. (n = 2 or 3). d Plasmid-based orthogonal R-loop assay. Here, EGFPstop was activated by Cas9-independent off-target DNA editing. Data are presented as mean ±s.d. (n = 3). P value was determined by two-way ANOVA Sidak’s multiple comparisons test. NS, not significant. e Orthogonal R-loop assay overview on human genomic loci. f Cas9-independent off-target A•T-to-G•C editing frequencies detected by the orthogonal R-loop assay at each R-loop site with dSaCas9 and a SaCas9 sgRNA. Each R-loop was performed by co-transfection of ABE8e or GS-ABE8e, and a SpCas9 sgRNA targeting site ABE site1 with dSaCas9 and a SaCas9 sgRNA targeting R-loops 1–5, respectively. Amplicons of R-loops sites were analyzed by high-throughput sequencing. For all plots, bars represent mean ±s.d. of three independent biological replicates. g On-target base editing efficiencies for ABE8e and GS-ABE8e in HEK293T cells at ABE site 1 in HEK293T cells for the orthogonal R-loop assay. Amplicons of ABE site 1 were analyzed by Sanger sequencing. For all plots, dots represent individual biological replicates and bars represent mean ±s.d. of three independent biological replicates. Source data are provided as a Source Data file.