Catalytically active Cas9 mediates transcriptional interference to facilitate bacterial virulence

• Type of Material: Article
• Authors: Hannah K. Ratner, Emory University; Andres Escalera-Maurer, Max Planck Unit for the Science of Pathogens; Anaïs Le Rhun, Max Planck Unit for the Science of Pathogens; Siddharth Jaggavarapu, Emory University; Jessie E. Wozniak, Emory University; Emily K. Crispell, Emory University; Emmanuelle Charpentier, Max Planck Unit for the Science of Pathogens; David Weiss, Emory University
• Language: English
• Date: 2019-08-08
• Publisher: Elsevier (Cell Press)
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: Published by Elsevier Inc.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.molcel.2019.05.029
• Title of Journal or Parent Work: MOLECULAR CELL
• Volume: 75
• Issue: 3
• Grant/Funding Information: This work was supported by National Institutes of Health (NIH) grants U54-AI057157 from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense, as well as R01-AI110701 to David S. Weiss, who is also supported by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award. We also thank the Alexander von Humboldt Foundation, the German Research Foundation, and the Max Planck Society for financially supporting this research study in the Charpentier lab.

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 has a highly specific regulon of four genes which must be repressed for bacterial virulence. Regulation occurs through a 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.