CRISPR-Cas systems: new players in gene regulation and bacterial physiology - PubMed (original) (raw)

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CRISPR-Cas systems: new players in gene regulation and bacterial physiology

Timothy R Sampson et al. Front Cell Infect Microbiol. 2014.

Abstract

CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

Keywords: CRISPR-Cas; Cas9; Francisella novicida; bacterial pathogenesis; post-transcriptional regulation of gene expression.

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Figures

Figure 1

Figure 1

Function of the Type II CRISPR-Cas system in adaptive nucleic acid restriction. (A) Foreign DNA is recognized by Cas1 and Cas2 and is processed into a new spacer sequence (red) within the CRISPR array (Adaptation phase, blue). (B) To restrict foreign DNA, the CRISPR array is transcribed as a single transcript (pre-crRNA array) and matured into small targeting crRNAs in a process requiring RNase III and tracrRNA. The dsRNA complex of crRNA and tracrRNA is associated with Cas9 and the spacer sequence within the crRNA can hybridize to complementary DNA sequences. Cas9 then mediates cleavage of the targeted DNA downstream of the proto-spacer adjacent motif, or PAM, highlighted by the red circle (Effector phase, pink).

Figure 2

Figure 2

CRISPR-Cas-mediated gene regulation and innate immune evasion by F. novicida. (A) A dual-RNA complex consisting of the tracrRNA and scaRNA forms through interaction of a sequence identical to the CRISPR repeat (pink box). This dsRNA structure is associated with F. novicida Cas9 and allows the free portion of the tracrRNA to interact through a non-identity interaction with mRNA encoding the BLP (FTN_1103; blue). Subsequently, the stability of the BLP mRNA is altered, possibly via catalytic activity of Cas9 (cleavage event indicated by red triangle) or by an unknown RNase (red sector). (B) Following entry into host cells, TLR2 is capable of detecting BLPs present in the F. novicida envelope. This leads to activation of a proinflammatory cytokine response. (C) However, by upregulating CRISPR-Cas components after entry and while in the phagosome, F. novicida limits its BLP content and dampens the activation of TLR2, leading to decreased proinflammatory cytokine signaling.

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