The recent discovery that bacteria and archaea employ an RNA-guided DNA cleavage mechanism to defend themselves from invasive genetic elements offers an unprecedented opportunity for understanding fundamental microbial biology and for developing biotechnology tools. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode three types of mechanistically different RNA-guided DNA cleavage enzymes that degrade invasive DNA while avoiding self-DNA. Understanding the molecular mechanisms of how these distinct DNA cleavage enzymes control their activities has important implications in basic enzymology, antibiotics resistance epidemics, human microbiome research, and genome editing. The Li laboratory has identified and purified representative members of two major types (Types II and III) of CRISPR-Cas DNA cleavage enzymes and is poised to unveil novel molecular mechanisms as well as to develop useful tools. Though both types are RNA-guided and invader-specific, these nucleases have drastically different in enzyme composition and activation mechanisms. An integrated approach ranging from cell-based assays, to structural biology and to fundamental enzymology will be employed to compare and contrast the mode of DNA interference by these nucleases, leading to an understanding of how microbe impact human health and biosphere and to an ultimate goal of developing CRISPR-based technology. The Li laboratory has assembled a team of scientists with complementary expertise in microbiology, nucleic acid biochemistry, mammalian cell biology, X-ray crystallography, and high-throughput cryogenic electron microscopy, in order to maximize the impact while mitigating risks of the research. Relevance: The CRISPR elements are found in more than 40% bacteria and are critical to maintenance of the overall microbial environment. The frequent occurrence of CRISPR in medically important bacteria that include but not limited to Yersinia pestis, Mycobacterium tuberculosis, Haemophilus influenzae, Helicobacter pylori, Neisseria meningitides, Vibrio vulnificus, Staphylococcus aureus, Salmonella Typhi, Clostridium tetani, and human microbiome relates CRISPR directly to human health. A thorough understanding of the CRISPR immunity has important implications in eradicating virulence and creating new antimicrobial strategies. While one of the CRISPR enzymes, namely Cas9, has been repurposed to serve as a user-specified genome-editing tool with ever-increasing popularity, we are yet to unleash the full potential of the CRISPR-derived tools in biomedical applications. The proposed research is aimed at overcoming current limitations while expanding the capability.

Public Health Relevance

Prokaryotes and archaea, including many human pathogens, employ a small RNA-based mechanism to maintain environmental balance through exchange and destruction of genetic materials. Structural studies are proposed to study the biochemical mechanism of this pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM099604-05A1S1
Application #
9478390
Study Section
Program Officer
Bender, Michael T
Project Start
2012-05-01
Project End
2020-12-31
Budget Start
2017-01-01
Budget End
2017-12-31
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Florida State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
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Wu, Jian-jun; Li, Wenwei; Shao, Yaming et al. (2015) Inhibition of cGAS DNA Sensing by a Herpesvirus Virion Protein. Cell Host Microbe 18:333-44
Li, Hong (2015) Structural Principles of CRISPR RNA Processing. Structure 23:13-20
Tsui, Tsz Kin Martin; Li, Hong (2015) Structure Principles of CRISPR-Cas Surveillance and Effector Complexes. Annu Rev Biophys 44:229-55
Peng, Yu; Yu, Ge; Tian, Shaoxiong et al. (2014) Co-expression and co-purification of archaeal and eukaryal box C/D RNPs. PLoS One 9:e103096
Hale, Caryn R; Cocozaki, Alexis; Li, Hong et al. (2014) Target RNA capture and cleavage by the Cmr type III-B CRISPR-Cas effector complex. Genes Dev 28:2432-43

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