CRISPR/Cas9-mediated genomic insertion of functional genes into <i>Lactiplantibacillus plantarum</i> WCFS1

Genetic engineering of lactic acid bacteria, such as <i>Lactiplantibacillus plantarum,</i> has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of <i>Lactiplantibacillus plantarum</i>. Our system successfully knock-in four expression cassettes varying in length from ~800-1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of <i>L. plantarum</i> strains.