Fig. 1: Design and mechanisms of two orthogonal glycosylase-based base editors.

a Prototype versions of a deaminase-free glycosylase-based thymine base editor (gTBE) and a deaminase-free glycosylase-based cytosine base editor (gCBE). PAM, Protospacer adjacent motif. AP, apurinic/apyrimidinic sites. Star in magenta indicates the nick generated by nCas9. b Schematic diagram of potential pathway for T (or C) editing and outcomes. A glycosylase variant is designed to remove normal T or C, an nCas9-sgRNA complex creates an R-loop at the target site and nicks the non-edited strand, then the generated AP site is repaired by translesion synthesis (TLS) and/or DNA replication, leading to T or C editing. DSB, double-strand break. indel, insertion and deletion. c Schematic of various gTBE and gCBE candidate architectures. The bipartite nuclear localization signal (bpNLS) is shown in dark gray, linker in light gray, nCas9 in teal green, and UNG in light green. Note that Y156A (purple line) and N213D (red line) of UNG2 are equivalent to Y147A and N204D of UNG1, respectively. Δ1-88: 1-88 amino acids truncation of UNG2. d Percentage of EGFP⁺ cells for T editing activity evaluation of different gTBE variants using T-to-G reporter (n = 3 independent biological replicates). NT, non-target sgRNA. T: target sgRNA. e Percentage of EGFP⁺ cells for C editing activity evaluation of different gCBE variants using C-to-G reporter (n = 3 independent biological replicates). NT, non-target sgRNA. T: target sgRNA. f the orthogonality of gTBE and gCBE for base editing evaluated using two different reporters (n = 3 independent biological replicates). All values are presented as mean ± s.e.m. Source data are provided as a Source Data file. Panel (a) adapted from Tong et al.³ (copyright 2023).