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Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence

Hannah K Ratner1, Andrés Escalera-Maurer2, Anaïs Le Rhun3

  • 1Microbiology 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.

Molecular Cell
|July 2, 2019
PubMed

Insights

The CRISPR-Cas9 system in Francisella novicida represses bacterial virulence genes using a small RNA (scaRNA). This natural system offers insights into gene regulation and potential therapeutic applications.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • The CRISPR-Cas9 system is known for foreign DNA defense.
  • Francisella novicida possesses a CRISPR-Cas9 system with an additional regulatory role.
  • This system represses an endogenous immunostimulatory lipoprotein, impacting bacterial virulence.

Purpose of the Study:

  • To investigate the specificity and molecular mechanism of CRISPR-Cas9-mediated gene regulation in F. novicida.
  • To understand how Cas9 controls a specific set of genes essential for bacterial virulence.
  • To explore the role of non-canonical small RNA (scaRNA) and tracrRNA in this regulatory process.

Main Methods:

  • Analysis of the CRISPR-Cas9 regulon in F. novicida.
  • Investigation of protospacer adjacent motif (PAM)-dependent interactions.
  • Characterization of scaRNA and tracrRNA involvement in transcriptional repression.
  • Reprogramming scaRNA for targeted gene repression and cleavage.

Main Results:

  • Cas9 regulates a specific four-gene set crucial for F. novicida virulence.
  • Regulation depends on PAM-dependent Cas9 binding, scaRNA, and tracrRNA.
  • Limited scaRNA complementarity prevents DNA cleavage, enabling transcriptional interference.
  • Repurposed scaRNA can direct Cas9 to repress or cleave exogenous targets.

Conclusions:

  • Francisella novicida CRISPR-Cas9 has evolved a unique mechanism for transcriptional repression using scaRNA.
  • This natural system demonstrates Cas9's broader regulatory potential beyond DNA cleavage.
  • Understanding this mechanism provides insights into bacterial physiology and potential biotechnological applications.
Keywords:
CRISPRCas9Francisella novicidabacterial pathogenesisgene regulationtranscription

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