Exploiting Endogenous CRISPR-Cas9 System for Functional Engineering of Probiotic Lacticaseibacillus rhamnosus GG (LGG)

Abstract

Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas9 genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable precise deletions and insertions across multiple loci. Using this optimized and precise genome engineering method, we generated a {beta}-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex gut microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/634116v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@4c471eorg.highwire.dtl.DTLVardef@1d133f6org.highwire.dtl.DTLVardef@1aa7400org.highwire.dtl.DTLVardef@1a5d4dc_HPS_FORMAT_FIGEXP M_FIG C_FIG