biostudies-literatureHao WPriority Academic Program Development of Jiangsu Higher Education InstitutionsNational Natural Science Foundation of ChinaHigher Education Discipline Innovation ProjectNatural Science Foundation of Jiangsu Province14395-14409https://www.ebi.ac.uk/biostudies/studies/S-EPMC9749471biostudies-literatureUnknown13(48)The functionally evolved bacterial chassis is of great importance to manufacture a group of assorted high value-added chemicals, from small molecules to biologically active macromolecules. However, the current evolution frameworks are less efficienct in generating <i>in vivo</i> genomic diversification because of insufficient tunability, rendering limited evolution spacing for chassis. Here, an engineered genomic diversification platform (CRISPR-ABE8e-CDA-nCas9) leveraging a programmable dual-deaminases base editor was fabricated for rapidly evolving bacterial chassis. The dual-base editor was constructed by reprogramming the CRISPR array, nCas9, and cytidine and adenosine deaminase, enabling single or multiple base conversion at the genomic scale by simultaneous C-to-T and A-to-G conversion <i>in vivo</i>. Employing titration of the Cas-deaminase fusion protein, the platform enabled editing any pre-defined genomic loci with tunable conversion efficiency and editable window, generating a repertoire of mutants with highly diversified genomic sequences. Leveraging the genomic diversification platform, we successfully evolved the nisin-resistant capability of <i>Bacillus subtilis</i> through directed evolution of the subunit of lantibiotic ATP-binding cassette. Therefore, our work provides a portable and programmable genomic diversification platform, which is promising to expedite the fabrication of high-performance and robust bacterial chassis used in the development of biomanufacturing and biopharmaceuticals.Chemical scienceConstruction and application of an efficient dual-base editing platform for <i>Bacillus subtilis</i> evolution employing programmable base conversion.PMC9749471111-2-062187812532171420BK20181206Han LCui WHao WSuo FCheng ZZhou ZfalseConstruction and application of an efficient dual-base editing platform for <i>Bacillus subtilis</i> evolution employing programmable base conversion.The functionally evolved bacterial chassis is of great importance to manufacture a group of assorted high value-added chemicals, from small molecules to biologically active macromolecules. However, the current evolution frameworks are less efficienct in generating <i>in vivo</i> genomic diversification because of insufficient tunability, rendering limited evolution spacing for chassis. Here, an engineered genomic diversification platform (CRISPR-ABE8e-CDA-nCas9) leveraging a programmable dual-deaminases base editor was fabricated for rapidly evolving bacterial chassis. The dual-base editor was constructed by reprogramming the CRISPR array, nCas9, and cytidine and adenosine deaminase, enabling single or multiple base conversion at the genomic scale by simultaneous C-to-T and A-to-G conversion <i>in vivo</i>. Employing titration of the Cas-deaminase fusion protein, the platform enabled editing any pre-defined genomic loci with tunable conversion efficiency and editable window, generating a repertoire of mutants with highly diversified genomic sequences. Leveraging the genomic diversification platform, we successfully evolved the nisin-resistant capability of <i>Bacillus subtilis</i> through directed evolution of the subunit of lantibiotic ATP-binding cassette. Therefore, our work provides a portable and programmable genomic diversification platform, which is promising to expedite the fabrication of high-performance and robust bacterial chassis used in the development of biomanufacturing and biopharmaceuticals.2022-01-01T00:00:00Z2022 Dec2026-05-27T23:03:22.435Z2024-11-10T03:45:09.651ZS-EPMC97494713654515210.1039/d2sc05824c