Determining the Structure and Function of Newly Discovered Immune Systems

Utah State University, Logan UT

Investigators

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Abstract

Project Summary Gaining a functional understanding of newly discovered immune systems (e.g. newly discovered CRISPR systems) will lead to novel molecular tools for use in science and medicine is the overarching hypothesis of my research program. Over the last 5 years we have generated a large amount of data supporting this hypothesis. Our efforts uncovered a distinct CRISPR associated (Cas) enzyme called Cas12a2 that can be programmed to selectively kill cells upon specific recognition of RNA sequence (project 2), creating new possibilities for targeted cell therapies and gene editing enrichment. With another CRISPR system (Type IV), we discovered another distinct programmable activity that may be able to be repurposed to silence gene expression while keeping targeted nucleic acid intact (project 1). The vision of my research program over the next five years is that we will determine, in deep enough detail, how the Type IV-A system and SuCas12a2 function to repurpose them as tools while expanding our studies into related but unstudied immune systems. Our specific goals for project 1 include (i) determining the role of CasDinG N-terminal domain that is essential for immune system activity, (ii) exploring how Type IV systems could be used to silence gene expression in mammalian cells, and (iii) determining the structure and function of other Type IV subsystems (e.g. Type IV-B and IV-C). Our goals for project 2 include (i) defining the determinants of SuCas12a2 activation by solving structures of Cas12a2 bound to mismatched targets and distinct PFS sequences and performing kinetic assay with diverse substrates, (ii) determining the distinct biochemical activities of Cas12a2 orthologs, and (iii) determining structures of Cas12a2 orthologs. To better understand the biological function of these immune systems, we are using cell-based and biochemical assays and are determining structures of immune system components at all stages of immunity using structural methods (x-ray crystallography and cryo-electron microscopy). We aim to determine how these systems identify their targets, how they distinguish self from non-self, and how distinctions from other immune systems (protein domains and genes) impact their function. Knowledge of these foundational mechanisms and their distinctions from existing CRISPR-based tools will be critical for repurposing these immune systems into novel molecular tools.