Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome...

Area of Science:

• Microbiology
• Synthetic Biology
• Genetic Engineering

Background:

• Live biotherapeutic products (LBPs) engineered from probiotics are crucial for disease treatment.
• Limosilactobacillus reuteri is a promising probiotic for LBP development, but genetic manipulation is challenging due to long editing cycles.

Purpose of the Study:

• To identify and characterize an endogenous CRISPR-Cas9 system in L. reuteri for efficient genetic engineering.
• To establish a robust genome editing platform for L. reuteri to facilitate LBP development.

Main Methods:

• Identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03, including the endogenous Cas9 (LrCas9) and its broad PAM recognition (3'-NDR).
• Reprogrammed LrCas9 for gene deletion, point mutation, large fragment deletion, and gene integration.
• Screened 304 L. reuteri strains for endogenous endonuclease distribution.

Main Results:

• Achieved high efficiency in gene deletion (95.46%), point mutation (86.36%), and gene integration (73.9%).
• Successfully deleted a 40 kb fragment and integrated a 1743 bp fragment.
• Found programmable endonucleases in 98.36% of L. reuteri strains, indicating broad applicability.

Conclusions:

• Developed a novel, efficient genome editing system for L. reuteri based on endogenous CRISPR-Cas9.
• The LrCas9 system facilitates functional genetic studies and accelerates the construction of LBPs.
• This platform enables genetic manipulation in the majority of L. reuteri strains, advancing probiotic engineering.