Application of the Streptococcus pyogenes CRISPR/Cas9 system in Lacticaseibacillus paracasei CGMCC4691

Lacticaseibacillus paracasei is a food-grade lactic acid bacteria (LAB) that plays an important role in improving the human intestinal tract. However, effective gene modification tools are not reported in L. paracasei CGMCC4691. Here, we constructed clustered regularly interspaced short palindromic repeat (CRISPR) genetic editing tools by screening and optimizing promoters of Cas9 and sgRNA, respectively. To verify the availability of this system, single gene mutants (4691ΔAF91_08090 and 4691ΔAF91_05150) and double gene mutants (4691ΔAF91_08090ΔAF91_05150) were obtained, the editing efficiency was 54.1% and 90% after replacing P1 promoter, respectively. In addition, the addition of 5-fluorouracil (5-Fu) was lethal to wild strains compared to mutants, therefore, the gene function of mutants was verified by growth phenotype. The study realizes efficient application of the Cas9 system in L. paracasei CGMCC4691, provides a feasible optimization method for gene editing, and lays the foundation for the investigation of genetic function mechanism.