Genomic and epigenetic landscapes drive CRISPR-based genome editing inBifidobacterium

Abstract

Bifidobacterium 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 B. animalis subsp. lactis, 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 tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis 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.

Establishing genetic manipulation for novel strains of human gut bacteria

“…In order to use CRISPR-Cas in gene editing, existing bacterial CRISPR-Cas systems are identified, the spacer motifs and target gene sequences synthesised and integrated into a suitable plasmid, and then transformed into the target host bacterium where the guide RNA targets the required insertion site [111] . CRISPR-Cas gene editing has been used successfully to modify the fermentation products of Clostridium acetobutylicum from acetone to isopropanol [112] and to abolish tetracycline resistance in Bifidobacterium animalis subsp. lactis [113] .…”

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

“…Alternatively, the type V CRISPR/Cas12a system, characterized by a single crRNA and a thymine-rich protospacer adjacent motif (PAM), is attractive and has been increasingly applied in bacterial genome editing in recent years [20,22,24]. In addition, the type I CRISPR systems, comprising the Cas3 nuclease and multiple effector proteins, have been reprogrammed to edit specific genome regions in its native hosts initially, and further successfully employed in heterologous editing in other bacterium species lacking the natural CRISPR systems [25][26][27].…”

In situtargeted mutagenesis of gut bacteria

“…To achieve efficient editing without killing the target bacteria we turned to base editors. Base editors convert one base pair to another at a target locus without introducing a double-strand DNA break 24 and have successfully been used in a broad range of bacterial species [25][26][27][28][29][30][31][32] .…”