The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) system is a prokaryotic adaptive immune system that defends microbes from foreign invaders such as viruses. This immune system incorporates DNA from invading elements into the CRISPR locus, essentially generating an immunization record of past viral infection. The subsequent transcription and processing of the CRISPR locus generates small CRISPR RNAs (crRNAs) that are incorporated into a protein complex that mediates the destruction of nucleic acids based on sequence complementarity. Imperfect complementarity between the crRNA and its nucleic acid target can abrogate cleavage, but still recruit the protein complex in vivo. This recruitment can result in alternative functions such as gene regulation, but the prevalence of these non-canonical CRISPR-Cas functions has not been investigated in detail. In fact, many pathogens and important human microbiome constituents possess CRISPR-Cas systems with no known role and CRISPR arrays with no predicted targets, raising a question as to what their biological functions are. The objective of this project is to identify endogenous bacterial proteins that may modulate CRISPR-Cas activity and discover and characterize novel CRISPR-Cas functions. This research is an extension of my graduate work, where I identified the first examples of CRISPR-Cas interacting proteins, which are a diverse group of inhibitors that directly interact with different CRISPR-Cas components. This suggests that more endogenous interactors remain to be identified. Together, the potential for nucleic acid binding in the absence of cleavage and the existence of CRISPR-Cas interacting proteins, suggests that there may be entire classes of bacterial proteins that can modulate or redirect CRISPR-Cas function. To broaden our understanding of the roles for CRISPR- Cas, I will utilize proteomic techniques to identify endogenous CRISPR-Cas interactors and characterize their physiological relevance. I will also conduct bio-informatic analyses to identify CRISPR-Cas systems that appear functional in pathogenic organisms and screen them for activity. Given the sequence diversity of the crRNAs generated by a single CRISPR-Cas system, both canonical (i.e. foreign genome cleavage) and non-canonical (i.e. gene regulation) functions could be mediated concurrently and functional assays will be developed to test these possibilities. The recent engineering of CRISPR-Cas systems to provide genome editing and regulatory tools in eukaryotic cells (where these systems do not naturally exist) perfectly exemplify the possibilities that are intrinsic to an RNA-guided system. The possibility that bacteria naturally possess similar functions (i.e. CRISPR-Cas-mediated recruitment of a transcription factor) has not been investigated. Assessing the roles of these systems in many different organisms will enhance our understanding of not only CRISPR-Cas systems, but also of how bacterial pathogens defend their genomes and regulate vital cell processes.

Public Health Relevance

Bacteria have an adaptive immune system called CRISPR-Cas that serves to protect the cell from viral attack. The CRISPR-Cas system also has other purposes, such as endogenous gene regulation, but the breadth of these alternative roles and the actual function of CRISPR-Cas in most organisms in fact, is unclear. This application outlines experiments to understand the role(s) of CRISPR-Cas in pathogenic bacteria, which will enhance our understanding of the evolution of disease causing organisms and could lead to novel therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Early Independence Award (DP5)
Project #
5DP5OD021344-02
Application #
9150686
Study Section
Special Emphasis Panel (ZRG1-RPHB-W (53)R)
Program Officer
Basavappa, Ravi
Project Start
2015-09-25
Project End
2020-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
2
Fiscal Year
2016
Total Cost
$396,250
Indirect Cost
$146,250
Name
University of California San Francisco
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94118
van Houte, Stineke; Ekroth, Alice K E; Broniewski, Jenny M et al. (2016) The diversity-generating benefits of a prokaryotic adaptive immune system. Nature 532:385-8