Validation of CRISPR-Based Tools for Engineering Bacteroides Species As Living Therapeutics

The gut microbiota impact human health and disease progression by interacting with the immune system, thereby modulating immune activity and efficacy of immunotherapies. Recent research has focused on identifying gut bacteria that are associated with specific disease outcomes. These organisms can be engineered to express beneficial metabolic pathways, thereby generating living therapeutics. However, current tools do not enable effective genetic manipulation of most gut microbes. CRISPR-based tools are powerful systems that may be used in diverse bacterial species, enabling genome editing and gene expression control.

We are leveraging our expertise with CRISPR-based genome engineering to develop tools that enable rapid and efficient editing of diverse gut microbes into controllable living therapeutics. We are validating these tools in Bacteroides species since they are abundant within the human colon and have been shown to play an important role in immune development. CRISPR-based tools may overcome the major limitations of existing genome editing methods for these microbes.

We present proof-of-concept data demonstrating deployment of Cas9 in Bacteroides species, which enables genomic editing of these bacteria, significantly decreasing the time and resources required compared to established methods. Catalytically dead Cas9 (dCas9) binds tightly to targeted sites in the genome, enabling gene expression modulation and control over metabolic pathways. Here, we show that dCas9 modulates the metabolism of Bacteroides species that, when delivered to the gut, have the potential to induce an immunomodulatory effect. Finally, we have designed a replicative plasmid system that is stable and replicates across Bacteroides species from human isolates. This tool facilitates heterologous gene overexpression throughout this genus.

Overall, these data highlight CRISPR-based tools that offer a unique and robust approach enabling engineering of key functional pathways in a suitable bacterial species based on microbiome-immune interaction studies. CRISPR-based editing is an essential platform for the rapid development of engineered microbial therapeutics.