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CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

Published on: September 13, 2024

Metabolically-targeted dCas9 expression in bacteria.

Gregory M Pellegrino¹, Tyler S Browne¹, Keerthana Sharath¹

¹Schulich School of Medicine and Dentistry, Department of Biochemistry, London, Ontario N6A 5C1, Canada.

Nucleic Acids Research

|January 11, 2023

Summary

This summary is machine-generated.

Researchers developed a novel biosensor to control gene expression in specific gut bacteria. This method targets glucuronidase-producing bacteria, offering a precise way to manage microbial gene activity in complex environments.

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Area of Science:

Microbiology
Synthetic Biology
Genomics

Background:

Targeting specific bacterial species within complex microbiomes remains a challenge.
Glucuronidase-utilization enzymes (GUS) are implicated in the reactivation of toxic compounds in the human gut, including chemotherapy drugs like irinotecan.

Purpose of the Study:

To develop a method for restricting gene expression to specific bacterial species based on their metabolic activity.
To re-purpose the glucuronide-responsive GusR transcription factor as a biosensor for controlling dCas9 expression.

Main Methods:

Fused the Escherichia coli gusA regulatory region to the dCas9 gene (pGreg-dCas9).
Tested dCas9 induction by glucuronides in E. coli.
Conjugated pGreg-dCas9 to Gammaproteobacteria from human stool samples.
Utilized dCas9-sgRNAs targeting the gusA operon for gene down-regulation.

Main Results:

dCas9 expression was specifically induced by glucuronides.
pGreg-dCas9 conjugation enabled dCas9 expression restricted to GUS-positive bacteria.
dCas9-sgRNAs achieved a ~100-fold decrease in GusA activity by down-regulating gus operon transcription.

Conclusions:

Demonstrated a general strategy for ligand-dependent expression of genetic tools like dCas9 in diverse bacteria.
Repurposed bacterial transcription factors responsive to exogenous metabolites for precise control of gene expression.