The CRISPR-based technologies will clearly play an outsized role in improving our understanding of gene regulation. The CRISPR system has been employed for the enhancement (activation) or attenuation (repression) of gene expression in a more reliable manner as compared to previous gene engineering techniques. The innovative use of the CRISPR system has been implemented to modulate gene expression through the alteration of epigenetic modifiers or crucial non-coding RNAs (ncRNAs). Importantly, the CRISPR system is of utmost importance in delineating the role of non-coding RNAs (miRNAs, lncRNAs) which are often implicated in tumor progression and heterogeneity.
CRISPR system constitutes a flexible platform that has been used for monitoring the localization of endogenous genes or cell fate in general. This improved understanding should ultimately lead to better predictive interpretation of patient germline and cancer genome sequences, a crucial goal for the future of precision medicine. Nonetheless, the CRISPR field is young, and it is exciting to imagine a future in which predictive models of gene regulation can guide genome editing in patients to treat a panoply of diseases. CRISPR-Cas9 gene editing has revolutionized our ability to alter genome sequence, and CRISPR-Cas9-based assays have already begun to contribute to new paradigms of gene regulation.
The most conclusive example of gene regulation by a CRISPR-Cas system comes from a study of the intracellular pathogen Francisella novicida. Scientists 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: tracrRNA and scaRNA (small CRISPR-Cas-associated RNA), to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP), which induces an inflammatory response. 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.
Fig 1. CRISPR-Cas mediated gene regulation and innate immune evasion by F. novicida.
Utilizing Cas9, tracrRNA, and scaRNA as regulators, F. novicida represses the expression of the targeted BLP, significantly lowering overall BLP levels in its envelope by roughly 2-fold. This allows the pathogen to effectively dampen TLR2 activation, facilitating its survival within the host. A dual-RNA complex consisting of the tracrRNA and scaRNA forms through interaction of a sequence identical to the CRISPR repeat. 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. Subsequently, the stability of the BLP mRNA is altered, possibly via catalytic activity of Cas9 or by an unknown RNase.
By upregulating CRISPR-Cas components after entry into host cells and while in the phagosome, F. novicida limits its BLP content and dampens the activation of TLR2, leading to decreased proinﬂammatory cytokine signaling. In the absence of this CRISPR-Cas mediated regulation, F. novicida elicits a significant TLR2-dependent inflammatory response, as revealed by the fact that cas9, tracrRNA, and scaRNA deletion mutants induce a much greater inflammatory response than wild-type bacteria. In addition, cas9, tracrRNA, and scaRNA deletion mutants are unable to induce a lethal infection of mice, further emphasizing their importance as regulators of virulence in F. novicida.
In addition to Cas9's role as a virulence factor in the bacterial pathogens F. novicida and N. meningitidis, CRISPR-Cas systems in other bacteria have been identified as having potential roles in virulence as well. Cas2, present within a Type II CRISPR system, is important for the ability of Legionella pneumophila to replicate within amoebae. while Type II CRISPR-Cas systems have similar genetic architectures, different species may have co-opted alternative components for functions distinct from defense against foreign nucleic acids. Type I CRISPR systems have also been implicated in aspects of bacterial physiology beyond their now canonical function in foreign nucleic acid defense. The Type I CRISPR system in Pseudomonas aeruginosa has been shown to play a role in modulating the production of biofilms.
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