Construction and application of an efficient dual-base editing platform for Bacillus subtilis evolution employing...

Area of Science:

• Synthetic Biology
• Genomics
• Biotechnology

Background:

• Bacterial chassis are crucial for producing valuable chemicals and biomolecules.
• Existing genomic diversification methods lack tunability, limiting chassis evolution.
• Efficiently generating diverse bacterial genomes is essential for biomanufacturing.

Purpose of the Study:

• To engineer a novel platform for rapid and tunable genomic diversification in bacteria.
• To overcome limitations of current evolution frameworks in bacterial chassis development.
• To create a programmable tool for generating diverse bacterial mutants.

Main Methods:

• Fabrication of an engineered genomic diversification platform using CRISPR-ABE8e-CDA-nCas9.
• Construction of a dual-base editor by reprogramming CRISPR array, nCas9, and cytidine/adenosine deaminases.
• Utilizing titration of Cas-deaminase fusion protein for tunable editing efficiency and genomic loci targeting.

Main Results:

• The platform enables simultaneous C-to-T and A-to-G base conversion in vivo at the genomic scale.
• Tunable conversion efficiency and editable windows allow for precise genomic modifications.
• Successfully evolved nisin-resistant Bacillus subtilis via directed evolution of a lantibiotic ATP-binding cassette subunit.

Conclusions:

• The developed platform offers a portable and programmable solution for bacterial chassis evolution.
• This technology accelerates the creation of high-performance bacterial chassis for biomanufacturing and biopharmaceuticals.
• The engineered system expands possibilities in microbial strain development and industrial biotechnology.