MicroDFBEST: A dCas12b-derived dual-function base editor with programmable editing characteristics for microbial genetic engineering

Base editors (BEs) enable precise genome editing, but their use in microbes remains limited by restricted mutagenesis capabilities and narrow editing windows. Here, we reported MicroDFBEST, a novel dual-function base editor (DFBE) for microbes, by fusing the high-activity deaminases evoCDA1 and TadA9 with nuclease-deficient Cas12b from Bacillus hisashii (dBhCas12b). This engineered system enables simultaneous C-to-T and A-to-G editing within a 26–33 nt window, the broadest range reported for microbial DFBEs. The editing characteristics of MicroDFBEST can be easily adjusted by changing fusion protein expression and editing generations to create diverse mutant libraries. We show that the MicroDFBEST system enables both flexible gene expression modulation via random promoter (PylbP) diversification and targeted protein evolution through mutational hotspot scanning in native genomic contexts. This study offers a versatile platform enabling in situ gene regulation (e.g., biosynthetic gene clusters activation) and protein evolution (e.g., chassis optimization), with broad synthetic biology utility.