Fig. 2: Protein engineering and evolution of gTBEs.

a Schematic diagram of mutagenesis and screening strategy for the engineered gTBE. The EGFP reporter plasmids were transiently co-transfected into cultured cells along with the gTBE plasmids, and the fluorescence intensity of EGFP was detected with flow cytometry. ΔNTD: N-terminal domain (NTD) truncation of UNG. b Left, the selected residues (shown as surface) for mutagenesis nearby the catalytic site pocket of human UNG-DNA complex (PDB entry 1EMH²⁴), in which dΨU was mutated to T in the DNA (dT). I150-L179 are highlighted in cyan, L210-T217 in magenta, A258-K261 in orange. Right, location of the effective residues in gTBEv3 variant shown as spheres in red on the three-dimensional structure. c Gradual improvement of EGFP activation for each gTBE variants (n = 3 independent biological replicates). WT, wild-type UNG2Δ88. dead, catalytically inactive UNG2Δ88 (carrying D154N and H277N mutations, equivalent to D145N and H268N of UNG1)⁶⁰. d Frequencies of T base editing outcomes (left) and indels (right) with different gTBE variants at the edited T5 position in site 9 (CLYBL gene) in transfected HEK293T cells by target deep sequencing (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).