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Research

Bacteria and archaea are the most abundant organisms on Earth and are connected to myriad aspects of our lives, from helping make the food we eat, to impacting our health and well-being, to shaping the environment we live in. Like us, they also face the threat of infection by their viruses, which are called phages. As a result, microbes have evolved diverse immune systems to protect themselves. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) loci are a class of prokaryotic adaptive immune systems that use RNA-guided effector complexes to defend against phages and other mobile genetic elements. Our lab aims to understand the diverse mechanisms by which CRISPR-Cas systems work, and use these insights to engineer new tools for biotechnology and medicine. We plan to employ versatile approaches, including biochemistry, molecular engineering, molecular, cell, and structural biology, to achieve these aims.

Artist's depiction of tandem CRISPR nucleases detecting RNA and generating a fluorescent signal in the FIND-IT assay
Depiction of the Fast Integrated Nuclease Detection in Tandem (FIND-IT) assay in action. Cas13a (yellow) recognizes target RNA and releases the activator molecule (orange), which in turn supercharges Csm6 (gray) to cleave and release the fluorescent reporter (bright green). This leads to generation of a fluorescent signal that can then be detected by a sensor. Artwork by Dr. Margaret Liu (University of Illinois-Chicago).
Exploring the diverse biology of prokaryotic immune systems

CRISPR-Cas systems are perhaps best known for their applications in genome engineering, in which programmable Class 2 CRISPR effectors, like Type II Cas9 and Type V Cas12, precisely edit DNA in a variety of cell types and organisms. However, Class 1 CRISPR systems, which include Types I, III, and IV, are far more abundant and widespread in nature. During my postdoc, I defined the nucleic acid targeting preference of a Class 1 CRISPR complex called Type III CRISPR Csm. Csm can degrade RNA and DNA, as well as produce signaling molecules that have the potential to activate many different downstream effector proteins, including nucleases, proteases, membrane proteins, and transcription factors. How these work and their role in immunity is not well understood. We aim to explore the diverse functions, mechanisms, and components of the Type III CRISPR signaling pathway. This could reveal entirely new layers of biology, redefining CRISPR as being much more than a pair of molecular scissors. It would also lay the foundation for developing novel tools for cellular engineering.

Tandem CRISPR nucleases recognize target RNA and amplify the signal for fluorescent detection.
Developing new technologies for nucleic acid detection and disease diagnostics

Conventional PCR-based methods for viral RNA detection are highly sensitive, but lack the simplicity and speed to be easily utilized for point-of-care diagnostics. Type III and VI CRISPR-associated (Cas) nucleases offer an attractive alternative, given their ability to directly bind and recognize RNA sequences. We previously showed that by deploying two unrelated Cas nucleases in tandem, we could detect as few as ~31 molecules of SARS-CoV-2 genomic RNA per microliter, a sensitivity that would be relevant for community surveillance of COVID-19. We also adapted this “FIND-IT” (Fast Integrated Nuclease Detection in Tandem) assay to function in a compact microfluidic device, illustrating its feasibility within a point-of-care diagnostic workflow. We aim to better understand and engineer this tandem nuclease chemistry for improved sensitivity, speed, and versatility of nucleic acid detection. We are also interested in adapting this approach for a broad range of diseases, i.e. cancer and infectious disease, and research applications.