Triple Base Editor Diversifies Three Nucleotides at Once

The editor, smACGmax, combines a Cas9 nickase with engineered adenine and cytosine deaminases and two alkyladenine DNA glycosylases. Recruiting base-excision repair produced transitions and transversions at all three nucleotides without creating programmed double-strand breaks.

Across endogenous sites in HEK293T cells, simultaneous triple-base editing ranged from 5.7% to 41%, and the number of resulting alleles was up to 3.2-fold higher than with a dual adenine–cytosine editor. Activity was also demonstrated in HeLa and HepG2 cells.

The researchers used 94 guide RNAs to diversify HBEGF, achieving substitutions at 94% of targeted amino-acid positions and identifying single and combinatorial variants that conferred resistance to diphtheria toxin. A second screen found more than 130 SF3B1 variants associated with altered splice-site selection, including known cancer-associated mutations.

Targeted assays generally showed comparable or fewer off-target edits than the dual editor, although whole-genome sequencing detected a slight increase in DNA substitutions. The experiments also revealed increased indel formation, and broader specificity studies are needed.

The study was led by Liang Chen at East China Normal University in Shanghai. It was published in Nature Communications on 27 July 2026.