Bacteria face a constant threat of being infected and killed by viruses, called bacteriophages, that are specially equipped to destroy them. In the Bondy-Denomy lab we are interested in the ways in which bacteria defend themselves from attack. We use a combination of genetic, molecular and biochemical approaches to characterize the arms race between bacteria and phages, with a goal to better understand microbial ecosystems. Furthermore, we hope to make discoveries that will be influential in combatting infectious disease and providing novel biotechnologies.
The CRISPR-Cas system was functionally characterized just ten years ago as a bacterial immune system that targets phages. Since then, there has been an explosion of interest in this system for its widespread presence in the microbial world as well as its facile programmability. This has formed the basis of a revolutionary gene editing technique, CRISPR-Cas9. In the lab, we are focused on studying CRISPR-Cas systems in their natural settings, asking what roles they perform for their host bacterium, how these systems are regulated and how phages fight back against CRISPR.
The battle between bacteria and phages has led to the evolution of multiple phage resistance mechanisms such as CRISPR-Cas and subsequent counter resistance mechanisms employed by the phage. While a graduate student in Alan Davidson’s lab, Joe discovered and characterized the first examples of phage-encoded proteins that inhibit CRISPR function, called anti-CRISPRs. These distinct proteins directly bind to and antagonize different CRISPR associated proteins, thus blocking phage targeting. We have now turned our attention to understanding why there are so many distinct anti-CRISPRs in a family of closely related phages and what the costs and benefits are to possessing different ones.
The early findings that a CRISPR array and the associated Cas genes could be transferred into a heterologous bacterial system and still be functional told us that, in general, these systems are autonomous. Further, with the successful transfer of some CRISPR-Cas systems into human cells and animals, it is quite clear that we understand the basic requirements for CRISPR function. What is poorly understood, however, is how CRISPR-Cas systems are regulated in their native hosts. What are the physiological cues that bacteria receive that can control CRISPR expression? We extensively use Pseudomonas aeruginosa as a model system for Type I CRISPR-Cas systems, but we know nothing about the factors that control its expression. This is an especially intriguing question due to a rich literature describing P. aeruginosa possessing more regulatory systems than most bacteria, presumably to equip it for its ‘generalist’ lifestyle, being both a ubiquitous microbe in the environment and a highly drug resistant opportunistic human pathogen.
CRISPR-Cas alternative functions
In addition to ‘canonical’ CRISPR function, we are also exploring other roles that CRISPR-Cas systems might fulfill for their host bacteria such as endogenous gene regulation and stress sensing. Both prokaryotes and eukaryotes use small RNA pathways to regulate endogenous gene expression. Thus we are currently investigating whether this is also a function for CRISPR, beyond simply targeting invading genetic parasites. This would have broad applications in understanding microbial pathogenesis and evolution and could lead to novel drug targets in the fight against antibiotic resistant pathogens.
Characterization of new CRISPR-Cas systems
We are always on the look out for microbes possessing new CRISPR-Cas systems that are yet to be characterized or studied. Approximately 40% of bacteria have CRISPR-Cas systems but we rely heavily on a small number of model organisms to inform our knowledge of CRISPR-Cas function and mechanism. With each new model CRISPR system, incredible new things are learned not only about CRISPR function but also what genetic insults that microbe sees in its natural environment. The spacers in a CRISPR array provide an unprecedented sequence specific tool to understand the natural ecology of a microbe by determining what it uses its CRISPR system for.