Genomic and epigenetic landscapes drive CRISPR-based genome editing in <i>Bifidobacterium</i>.

Abstract

<i>Bifidobacterium</i> is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in <i>B. animalis</i> subsp. <i>lactis,</i> demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in <i>tetW</i> to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple <i>B. lactis</i> strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among <i>B. lactis</i> strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to <i>Bifidobacterium longum</i> subsp. <i>infantis</i> to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.