Enhanced editing of <i>Bifidobacterium lactis</i> using the endogenous Type I-G CRISPR-Cas system

Diverse <i>Bifidobacterium animalis</i> subsp. <i>lactis</i> strains are widely used as commercial probiotics. While proof-of-concept studies have shown that some strains can be edited using several CRISPR-Cas approaches, this species remains difficult to engineer, hindering functional genomic studies to establish their molecular mode of action and enhance their probiotic functionalities. Here, we show that >95% of available <i>B. lactis</i> genomes harbor a conserved Type I-G CRISPR-Cas system, which we leverage to develop and validate a broadly applicable genome editing framework. We redesigned backbone plasmids with different replicons and antibiotic resistance markers and evaluated performance across six commercial strains for transformation efficiency. A vector carrying the pBC1 origin coupled with a chloramphenicol resistance marker improved transformation in most strains. Using synthetic CRISPR arrays with self-targeting spacers in combination with homologous editing templates, we tested multiple spacers and evaluated short (600 bp) versus long (1,000 bp) homology arms. To demonstrate applicability, we generated knockouts in three glycoside hydrolases within the Balac 1593-1601 cluster, readily cured editing plasmids in non-selective medium, and performed iterative genome editing. Growth phenotyping across carbohydrates confirmed that the GH36 α-galactosidase Balac 1601 knockout abolished melibiose and raffinose utilization, and that deletions within Balac 1596 and Balac 1593 carbohydrate hydrolases produced non-canonical phenotypes, suggestive of a modulatory role associated with shift in carbon use and compensation by other pathways. These results establish a practical toolkit for editing diverse <i>B. lactis</i> strains, unravel the genomics underlying probiotic attributes, and provide a blueprint for genome engineering in other non-model probiotic bacteria.IMPORTANCE<i>Bifidobacterium animalis</i> subsp. <i>lactis</i> strains are prominent probiotics widely formulated in foods and dietary supplements, yet remain difficult to engineer, limiting efforts to connect genes to probiotic traits and to build strains with enhanced functions. Here, we harness the native Type I-G CRISPR Cas system to enable genome editing across commercial <i>B. lactis</i> strains by optimizing a compact plasmid backbone, testing multiple spacers to achieve efficient editing, and selecting homology arms of the appropriate length for recombination. With this framework, we generate knockouts at multiple, functionally distinct loci, demonstrating target-agnostic applicability, and we cure the CRISPR-editing vectors efficiently, enabling sequential edits. This toolkit enables systematic genotype-to-phenotype mapping in <i>B. lactis</i> and provides a practical framework for strain improvement in organisms of industrial relevance.