Exploiting the Endogenous Type II-A CRISPR-Cas System for Functional Engineering of Probiotic Lacticaseibacillus...

Area of Science:

• Microbiology
• Genetic Engineering
• Probiotics

Background:

• Lacticaseibacillus rhamnosus GG (LGG) is a well-researched probiotic with broad applications.
• Limited genome editing tools hinder the functional enhancement and versatility of LGG.
• Efficient and precise genetic engineering is crucial for developing advanced probiotic strains.

Purpose of the Study:

• To establish an endogenous CRISPR-Cas genome editing workflow for Lacticaseibacillus rhamnosus GG.
• To enable precise genetic modifications for functional strain construction in LGG.
• To facilitate the development of next-generation probiotics for diverse applications.

Main Methods:

• Developed and optimized an endogenous type II-A CRISPR-Cas system for LGG.
• Utilized a plasmid interference assay and single-nucleotide substitutions to define the PAM sequence (5'-NGAAA-3').
• Employed synthetic sgRNA cassettes and homology-directed repair donors for targeted gene deletions and insertions.

Main Results:

• Achieved practical genome editing efficiencies (11.1-25.0%) in recovered transformants for routine strain construction.
• Successfully generated a β-glucuronidase (GUS)-expressing LGG strain for microbial community tracking.
• Demonstrated the precision and applicability of the developed genome engineering method.

Conclusions:

• The developed CRISPR-Cas workflow overcomes previous limitations in LGG genetic engineering.
• This advancement expands the available CRISPR toolkit for probiotic research.
• Provides versatile strategies for designing next-generation probiotics for food biotechnology and therapeutics.