Utilization of Native CRISPR-Cas9 System for Expression of Glucagon-like Peptide-1 in Lacticaseibacillus paracasei.

Type 2 diabetes is one of the main causes of cardiovascular diseases, kidney diseases, and visual impairments, posing a global healthcare challenge. The current treatment of this disease, involving glucagon-like peptide-1 (GLP-1), is faced with problems such as frequent injections and plasmid instability. In this study, we used the native clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system of Lacticaseibacillus paracasei to develop a novel, genetically stable, and orally administrable strain expressing human GLP-1. Integration and subsequent expression of glp-1 gene were confirmed by genomic sequencing, qPCR, and Nano LC-MS. The engineered strain demonstrated stable genomic integration and sustained high-level expression of GLP-1 over multiple generations. This innovative approach provides a promising strategy for the oral delivery of therapeutic peptides, potentially enhancing patient compliance and improving the treatment of diabetes and other chronic diseases requiring peptide-based therapies.