Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei

Area of Science:

• Microbiology
• Genetic Engineering
• Synthetic Biology

Background:

• Lacticaseibacillus paracasei is a safe, health-promoting microbe widely used in food and pharmaceuticals.
• Understanding the genetic basis of L. paracasei's beneficial traits is limited by a lack of effective genetic manipulation tools.
• While type II CRISPR-Cas9 systems are common in lactobacilli, they have not been developed for genetic modification.

Purpose of the Study:

• To establish the first endogenous CRISPR-Cas9 genome-editing system in Lacticaseibacillus paracasei.
• To develop a precise and efficient genetic tool for L. paracasei engineering.
• To enable advancements in the genetics and biotechnology of lactobacilli.

Main Methods:

• Reprogramming the native CRISPR-Cas9 system using a validated protospacer adjacent motif (PAM) and customized single guide RNA (sgRNA) expression cassette.
• Developing an all-in-one vector integrating a controllable plasmid-targeting sgRNA expression module.
• Implementing gene deletion, chromosomal insertion, nucleotide substitution, large fragment deletion, and simultaneous multi-gene deletion strategies.

Main Results:

• Achieved over 90% efficiency for gene deletion and chromosomal insertion.
• Reached ≥50% efficiency for nucleotide substitution.
• Successfully deleted large genomic fragments (5-10 kb) and multiple genes at distal loci, firsts for lactobacilli.
• Demonstrated high efficiency (>90%) for gene deletion and plasmid curing using the all-in-one vector.

Conclusions:

• The developed endogenous CRISPR-Cas9 system provides a convenient and precise genetic tool for Lacticaseibacillus paracasei.
• This breakthrough facilitates the genetic engineering of L. paracasei, paving the way for further research and applications.
• The study significantly contributes to the broader field of lactobacilli genetics and engineering.