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

• CRISPR/Cas9 system was screened and successfully applied to Lacticaseibacillus paracasei genome knockout. • Among the four promoters screened from Lacticaseibacillus paracasei , the P1 promoter had higher transcriptional activity in both CRISPR systems. • Multigene editing mediated by CRISPR/Cas9 was achieved to knockout upp gene in consecutive steps. • The key genes associated with upp function were identified. 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 (4691Δ AF91_08090 and 4691Δ AF91_05150 ) and double gene mutants (4691Δ AF91_08090 Δ AF91_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.