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Mol Cell. 2019 Aug 8;75(3):498-510.e5. doi: 10.1016/j.molcel.2019.05.029. Epub 2019 Jun 27.

Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.

Author information

1
Microbiology and Molecular Genetics Program, Emory University, Atlanta, GA 30329, USA; Emory Vaccine Center, Emory University, Atlanta, GA 30329, USA; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA.
2
Max Planck Unit for the Science of Pathogens, 10117 Berlin, Germany; Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, 38124 Braunschweig, Germany; Institute for Biology, Humboldt University, 10115 Berlin, Germany.
3
Max Planck Unit for the Science of Pathogens, 10117 Berlin, Germany; Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, 38124 Braunschweig, Germany.
4
Emory Vaccine Center, Emory University, Atlanta, GA 30329, USA; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA; Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30329, USA.
5
Microbiology and Molecular Genetics Program, Emory University, Atlanta, GA 30329, USA; Emory Vaccine Center, Emory University, Atlanta, GA 30329, USA; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30329, USA; Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30329, USA. Electronic address: david.weiss@emory.edu.

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.

KEYWORDS:

CRISPR; Cas9; Francisella novicida; bacterial pathogenesis; gene regulation; transcription

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