Abstract
In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.
Highlights
CRISPR-Cas systems are prokaryotic adaptive immune systems that restrict infection by potentially harmful foreign genetic elements (Barrangou et al, 2007; Marraffini and Sontheimer, 2008)
F. novicida Cas9 (FnoCas9) Has a Highly Specific Regulon To identify regulatory targets of FnoCas9, we performed a genome-wide analysis of mRNA levels in a cas9 deletion mutant compared to wild-type (WT) F. novicida
4 out of 1,782 genes (0.22%) were significantly upregulated in the cas9 mutant by more than 2-fold, including FTN_1103 (1103) encoding a bacterial lipoprotein, which we previously demonstrated to be regulated by FnoCas9 (Figures 1A, S1A, and S1C–S1F) (Jones et al, 2012; Sampson et al, 2013)
Summary
CRISPR-Cas systems are prokaryotic adaptive immune systems that restrict infection by potentially harmful foreign genetic elements (Barrangou et al, 2007; Marraffini and Sontheimer, 2008). The other is tracrRNA (trans-activating crRNA), a small RNA transcribed from the CRISPR-Cas locus that contains an inverted sequence with complementarity to the repeat sequence conserved in each crRNA derived from a CRISPR array (Deltcheva et al, 2011). Another portion of the crRNA, the spacer sequence, is often complementary to an exogenous DNA target (Bolotin et al, 2005; Mojica et al, 2005; Pourcel et al, 2005). Upon infection with a nucleic acid, the crRNA spacer binds to the complementary sequence on the incoming DNA (the protospacer), leading Cas to cleave the DNA target, resulting in a double-strand break (Gasiunas et al, 2012; Jinek et al, 2012)
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