LAUSR

Significance Genome engineering in Bifidobacterium remains challenging, despite successful CRISPR deployment in other bacteria, hindering the comprehensive understanding of the molecular mechanisms of health-promoting effects. In this study, we adapted existing CRISPR effectors and editing strategies to generate a variety of editing outcomes in B. animalis subsp. lactis. We also showed that the editing efficiency and genome accessibility are shaped by the genomic and epigenetic landscapes, despite their monomorphic genomes. Together, these findings emphasize that strain-to-strain variation can impact the deployment of genome editing and lead to phenotypic and functional differences. This study expands the genome editing platform of Bifidobacterium to enhance their probiotic efficacy and opens opportunities for the engineering of bacterial biotherapeutics.