It has recently been discovered that prokaryotes can acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clusters of regularly interspaced short palindromic repeats (CRISPR's). These repeats are then transcribed and processed into small guide RNA's that are used to direct the destruction of foreign nucleic acid. This mechanism has many parallels with eukaryotic RNA interference but the proteins that are associated with the CRISPR response are evolutionarily unrelated to their eukaryotic counterparts. Our long-term goal is to understand the biochemical and structural basis of CRISPR-mediated resistance in prokaryotes. The objective here is to determine the mechanisms used to produce guide RNA's from CRISPR transcripts. Despite recent advances, understanding of these mechanisms is rudimentary. Our objective will be achieved through biochemical, structural and cell based analyses of CRISPR transcripts and the CRISPR-associated (cas) proteins. We hypothesize that in all prokaryotes this process will require the specific and sequential action of multiple cas proteins and that the fundamental mechanism will be conserved. Successful completion of the proposed studies is significant because it will increase our understanding of bacterial resistance to viruses and plasmids. Both of these genetic elements play important roles in the genetics of pathogenic bacteria.

Public Health Relevance

The proposed research is relevant to public health because it will increase our understanding of the interplay between pathogenic bacteria and mobile genetic elements, such as viruses and plasmids. This interplay is instrumental to the acquisition of antibiotic resistance, and evolution, of pathogen bacteria. Thus, the proposed research is relevant to the NIH's goals of improving the control of disease, enhancing human health and advancing our understanding of biological systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097330-04
Application #
8708123
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Bender, Michael T
Project Start
2011-08-15
Project End
2016-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia et al. (2016) Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation. Nucleic Acids Res 44:10849-10861
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
Ramachandran, Anita; Bailey, Scott (2016) Memory Upgrade: Insights into Primed Adaptation by CRISPR-Cas Immune Systems. Mol Cell 64:641-642
Estrella, Michael A; Kuo, Fang-Ting; Bailey, Scott (2016) RNA-activated DNA cleavage by the Type III-B CRISPR-Cas effector complex. Genes Dev 30:460-70
Mallon, John; Bailey, Scott (2016) A molecular arms race: new insights into anti-CRISPR mechanisms. Nat Struct Mol Biol 23:765-6
Chen, Hongfan; Bailey, Scott (2016) Structural biology. Cas9, poised for DNA cleavage. Science 351:811-2
van Erp, Paul B G; Jackson, Ryan N; Carter, Joshua et al. (2015) Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. Nucleic Acids Res 43:8381-91
Mulepati, Sabin; Héroux, Annie; Bailey, Scott (2014) Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science 345:1479-84
Chen, Hongfan; Choi, Jihoon; Bailey, Scott (2014) Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem 289:13284-94
Mulepati, Sabin; Bailey, Scott (2013) In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target. J Biol Chem 288:22184-92

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