Development and implementation of a Type I-C CRISPR-based programmable repression system for Neisseria gonorrhoeae

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are prokaryotic adaptive immune mechanisms.
• Neisseria gonorrhoeae (Gc) lacks an endogenous CRISPR system, hindering genetic manipulation.
• Neisseria lactamica possesses a functional Type I-C CRISPR-Cas system.

Purpose of the Study:

• To develop a programmable and inducible CRISPR interference (CRISPRi) platform for gene repression in Gc.
• To establish a tool for interrogating essential and non-essential genes in Gc.
• To facilitate the study of Gc physiology, pathogenesis, and antimicrobial development.

Main Methods:

• Engineered an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible CRISPRi system from N. lactamica Type I-C CRISPR, lacking the Cas3 nuclease.
• Targeted specific genes, including the opaD gene and the entire opa gene family, for transcriptional repression.
• Assessed gene and protein expression, bacterial phenotypes, and reversibility of repression upon IPTG removal.

Main Results:

• Demonstrated successful downregulation of opaD gene and protein expression, impacting bacterial function.
• Showed effective knockdown of all 11 opa genes using a five-spacer CRISPR array.
• Confirmed reversible gene repression and conditional reduction of essential gene expression.

Conclusions:

• The developed Type I-C CRISPRi system provides a powerful tool for locus-specific transcriptional repression in Gc.
• This system enables the functional characterization of essential genes that are otherwise difficult to study.
• The CRISPRi platform holds significant potential for advancing Gc research and aiding in the development of novel therapeutics.