This project is intended to provide a detailed understanding of a recently discovered prokaryotic adaptive immune system known as CRISPR (clusters of regularly interspaced short palindromic repeats). CRISPR drives adaptation to harmful invading nucleic acids - such as conjugative plasmids, transposable elements and phages - using an RNA-mediated interference (CRISPR interference) mechanism with fundamental similarities to our innate and adaptive immune responses. CRISPR-Cas defense systems have been identified in 88% of archaeal genomes and 39% of bacterial genomes thus far sequenced, including important human pathogens such as Campylobacter human jejuni, Clostridium botulinum, Escherichia coli, Listeria monocytogenes, Mycobacterium tuberculosis and Yersinia pestis. It has been shown to modulate the horizontal gene transfer and biofilm formation. Although the details of this defense mechanism remain to be determined, two distinct stages are recognized: (i) adaptation upon first exposure to the foreign nucleic acid whereby some combination of CRISPR-associated (Cas) proteins extracts recognizable features from the genomes of viruses (bacteriophages) and plasmids as protospacers that are subsequently incorporated as spacers at the 5'end of genomic CRISPR loci;and (ii) interference upon re-exposure to the same nucleic acid whereby a ribonucleoprotein complex comprised of small guide RNAs (crRNA) derived from genomic CRISPRs and different Cas proteins targets foreign nucleic acids for destruction. The lack of information on the molecular and structural properties of the Cas proteins and complexes severely impedes progress in the study of CRISPR mediated bacterial immunity. The proposed research is based on the successful structure determination of several important Cas proteins and the successful reconstitution of the Type I-C Cascade complex from B. halodurans. In this proposal, we propose experiments to understand the CRISPR interference mechanism in Type I-C CRISPR-Cas system. We build upon strong preliminary data to (1) characterize the structure-function of individual components of the Type I-C Cascade, (2) establish function assays and determine the EM and crystal structure of the intact I-C Cascade, and (3) characterize the structure-function of the Cascade-interacting protein Cas3, an essential factor in all Type I CRISPR-Cas systems. Our findings will serve to reveal the common theme and mechanistic diversity among different CRISPR-Cas systems.

Public Health Relevance

The described structure-function studies of CRISPR interference system in bacteria will contribute to the development of strategies to manipulate the CRISPR-Cas operon to control the proliferation and virulence of microbial pathogens such as Campylobacter human jejuni, Clostridium botulinum, Escherichia coli, Listeria monocytogenes, Mycobacterium tuberculosis and Yersinia pestis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM102543-01A1
Application #
8505857
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2013-07-01
Project End
2017-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
$287,911
Indirect Cost
$104,294
Name
Cornell University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Xiao, Yibei; Luo, Min; Dolan, Adam E et al. (2018) Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science 361:
Xiao, Yibei; Luo, Min; Hayes, Robert P et al. (2017) Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System. Cell 170:48-60.e11
Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun et al. (2017) How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration. Nature 550:137-141
Hayes, Robert P; Xiao, Yibei; Ding, Fran et al. (2016) Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli. Nature 530:499-503
Mizrachi, Dario; Chen, Yujie; Liu, Jiayan et al. (2015) Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy. Nat Commun 6:6826
Nam, Ki H; DeLisa, Matthew P; Ke, Ailong (2015) Characterizing Metal-Dependent Nucleases of CRISPR-Cas Prokaryotic Adaptive Immunity Systems. Methods Mol Biol 1311:265-76
Hayes, Robert P; Ke, Ailong (2015) One more piece down to solve the III-A CRISPR puzzle. J Mol Biol 427:228-30
Price, Ian R; Gaballa, Ahmed; Ding, Fang et al. (2015) Mn(2+)-sensing mechanisms of yybP-ykoY orphan riboswitches. Mol Cell 57:1110-1123
Grigg, Jason C; Ke, Ailong (2015) Structures of Large RNAs and RNA-Protein Complexes: Toward Structure Determination of Riboswitches. Methods Enzymol 558:213-232
Liu, Jun-Jie; Bratkowski, Matthew A; Liu, Xueqi et al. (2014) Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM. Nat Struct Mol Biol 21:95-102

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