Synthetic Approaches to Engineering Phage Transduction

Commensal microbes from the human gastrointestinal (GI) tract have been shown to directly impact human health. We can magnify their beneficial effects by editing the genomes of these organisms in order to control when and where specific metabolic pathways are activated, thereby creating controllable, living therapeutics. Current tools do not enable effective genetic manipulation of most gut microbes. DNA delivery into uncultivated bacteria remains a significant initial barrier to genetic manipulation of commensal bacteria. Bacteriophages, bacterial viruses that have evolved to deliver their DNA into bacteria, represent an exciting tool to overcome this obstacle. At Caribou Biosciences, Inc., we are developing a platform for phage manipulation to alter surface receptor specificity and enable plasmid delivery to specific bacterial targets.

Data is presented describing our efforts utilizing bioinformatics, DNA synthesis, NGS assays, and Cas9-based phage manipulation to modify DNA packaging and tropism interactions. The primary focus is on engineering phages in vitro through the use of cell-free systems (CFS). We engineer a phage genome with Cas9 RNPs to modify host range via CFS-based genetic complementation. We demonstrate packaging of Cas9-based tools in a CFS and delivery of those tools to a host microbe. These achievements, along with other ongoing work, will pave the way to significantly decrease the time required to successfully engineer bacteria isolated from the human GI tract.