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85 of 109 species shown to possess Cas9 homologues are known pathogens including N. meningitis and C. jejuni [3,9]. In addition, type-II CRISPR systems are predominately found in mammalian pathogens [10].These data showcase the breadth of CRISPR/Cas systems in increasing pathogenesis. Specifically, C. jejuni pathogenesis in Guillain–Barré syndrome has been linked to a CRISPR/Cas system that increases expression of Cst-II sialyltransferase, a necessary component in generating liposaccharide structures necessary for pathogenesis [10]. CRISPR gene inactivations significantly reduced virulence in Cst-II positive C. jejuni [10]. Additionally, a Cas9 deletion mutant of N. meningitis showed a lowered ability to infect and replicate in human epithelial cells, indicating a strong role for the CRISPR/Cas system in N. meningitis pathogenesis [3]. Therefore, investigating the role of the CRISPR system in down-regulating BLP expression in F. novicida will frame studies on more significant, biologically relevant species.
The canonical CRISPR/Cas system has recently been manipulated to edit genes of interest with high specificity in models including human, bacterial, yeast, rodent, and zebrafish cells [11]. With this specificity, CRISPR has solidified itself as a genome editing tool effective for multiple species [ 12-14]. In addition, the need for a crRNA and tracrRNA to be transcribed in vivo has been eliminated by combining the functions into a single, double-stranded, artificial guide RNA (gRNA) [11]. A similar approach could be attempted in an RNA-targeting CRISPR system, and increases the scope of possible genome expression targets. The significance of RNA targeting is clear when it is understood that targeting of DNA alone has limitati...

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...SPR upregulation. I expect the half-life of FTN_1103 in some of these knockout strains to be significantly increased. This will be interpreted as a specific endonuclease being recruited by the Cas9-tracrRNA-scaRNA-mRNA complex for degradation. Limitations: Some of these nucleases may have degenerate functionality. Failing to find increased FTN_1103 halflife, deletion combinations of these six endonucleases will be created and tested similarly. It is also possible that the endonuclease required is undescribed or otherwise not tested. If the deletion assays fail, protein crosslinking in vivo during phagosome infection will be performed in cells expressing myc-tagged Cas9. Cas9 will then be immunoprecipitated and the attached proteins will be separated and PAGE analyzed. The non Cas9 proteins will then be subjected to tandem mass spectrometry to identify endonucleases.
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