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

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Genome editing

Background:

• Existing base editors (BEs) have limitations in microbial applications, including restricted mutagenesis and narrow editing windows.
• Developing novel tools is crucial to enhance genome editing precision and versatility in microorganisms.

Purpose of the Study:

• To engineer a novel dual-function base editor (DFBE) for microbes, named MicroDFBEST.
• To expand the capabilities of base editing in microbial systems for diverse synthetic biology applications.

Main Methods:

• Fusing high-activity deaminases (evoCDA1 and TadA9) with a nuclease-deficient Cas12b (dBhCas12b).
• Utilizing MicroDFBEST for simultaneous C-to-T and A-to-G editing within a 26-33 nt window.
• Adjusting editing characteristics by modifying fusion protein expression and editing generations.

Main Results:

• Achieved the broadest editing window reported for microbial DFBEs.
• Demonstrated flexible gene expression modulation through promoter diversification (P_ylbP).
• Enabled targeted protein evolution via mutational hotspot scanning in native genomic contexts.

Conclusions:

• MicroDFBEST provides a versatile platform for in situ gene regulation, such as activating biosynthetic gene clusters.
• The system facilitates protein evolution, including chassis optimization, with broad synthetic biology utility.