Highly efficient CRISPR-mediated base editing for the gut Bacteroides spp. with pnCasBS-CBE

Area of Science:

• Microbiology
• Genetics
• Biotechnology

Background:

• Bacteroidales are dominant bacteria in the human gut microbiome.
• These bacteria hold therapeutic potential.
• Genetic tools for Bacteroidales are limited.

Purpose of the Study:

• To develop a novel base editing system for Bacteroidales.
• To expand the genetic toolkit for Bacteroides thetaiotaomicron.
• To enable functional genomic analysis in Bacteroidales species.

Main Methods:

• Construction of the pnCasBS-CBE system for C:G to T:A base editing.
• Application of the system for introducing mutations and stop codons in carbohydrate metabolism genes.
• Demonstration of multiplexed gene editing (up to four genes).
• Validation in four additional non-model gut Bacteroides species.
• Genome-wide SNP analysis for fidelity assessment.

Main Results:

• Efficient C:G to T:A base editing was achieved in Bacteroides thetaiotaomicron.
• Nonsynonymous mutations and stop codons were successfully introduced.
• Multiplexed editing of up to four genes was demonstrated in a single experiment.
• The system proved effective in four other Bacteroides species.
• High fidelity and applicability of the pnCasBS-CBE system were confirmed.

Conclusions:

• The pnCasBS-CBE system is a powerful tool for genome editing in Bacteroidales.
• This system significantly advances functional genomic analysis in gut Bacteroides.
• It provides a foundation for exploring therapeutic applications of Bacteroidales.