The recent CRISPR-Cas12b gene editing system, which recognizes DNA targets based on reprogrammed RNA, is increasingly being utilized in various fields. We developed an advanced base-editing technology that seamlessly integrates the TadA8e deaminase with an inactivated dBhCas12b (dead Bacillus hisashii Cas12b) module, enabling precise single-base DNA substitutions in human cell lines. Using the TadA8e-dBhCas12b module, we successfully induced single-base (A-to-G) substitutions at genomic target sites in HEK293FT cells, with an efficiency ranging from 3.96% ± 1.10%-25.78% ± 0.78%, with minimal unintended indel formation due to target DNA nicking. Notably, the editing window of the TadA8e-dBhCas12b module, spanning approximately positions 5-10, can be precisely modulated through the introduction of point mutations within the RuvC domain and the fusion of nonspecific DNA-binding domains, such as Sso7d. These modifications enable efficient base editing at target sites containing thymine-rich protospacer-adjacent motifs (PAMs). The optimized TadA8e-dBhCas12b platform therefore represents a complementary alternative to conventional CRISPR-Cas9-derived base editors and may broaden the range of targetable pathogenic variants, particularly those located within T-rich genomic contexts. Its DNA context-dependent editing window and expanded targeting capacity highlight its potential utility in both basic biological research and therapeutic genome editing.
