Genome Editing Using the Endogenous Type I-E CRISPR-Cas System in Lactobacillus paracasei ATCC334

Area of Science:

• Microbiology
• Genetics
• Biotechnology

Background:

• Lactobacillus paracasei ATCC334 is a beneficial bacterium with applications in food and health.
• Limited gene editing techniques hinder understanding and utilization of L. paracasei ATCC334.
• Efficient gene editing is crucial for unlocking the full potential of this probiotic strain.

Purpose of the Study:

• To explore and characterize the endogenous type I-E clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system in L. paracasei ATCC334.
• To establish and validate gene editing tools for L. paracasei ATCC334.
• To engineer L. paracasei ATCC334 for novel applications, such as nicotine degradation.

Main Methods:

• Bioinformatic analysis of the endogenous CRISPR-Cas system, including repeat, spacer, and leader sequences.
• Prediction and validation of the protospacer adjacent motif (PAM).
• Assessment of CRISPR-Cas system cutting activity and gene editing efficiency through plasmid and genome interference experiments.

Main Results:

• Identification and characterization of a type I-E CRISPR-Cas system in L. paracasei ATCC334.
• Successful validation of PAM recognition and CRISPR-Cas system activity.
• Demonstration of efficient gene knockout and gene integration using the developed gene editing tools.
• Engineering of an L. paracasei strain capable of degrading nicotine.

Conclusions:

• The study successfully established CRISPR-Cas based gene editing in L. paracasei ATCC334.
• This provides a powerful platform for genetic manipulation and functional studies of lactobacilli.
• The developed methods pave the way for engineering lactobacilli for diverse industrial and health applications.