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.
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.
|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|
|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|
|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|
|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; HÃ©roux, Annie; Bailey, Scott (2014) Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science 345:1479-84|
|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|
|Mulepati, Sabin; Orr, Amberly; Bailey, Scott (2012) Crystal structure of the largest subunit of a bacterial RNA-guided immune complex and its role in DNA target binding. J Biol Chem 287:22445-9|
|Mulepati, Sabin; Bailey, Scott (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem 286:31896-903|