Development and implementation of a Type I-C CRISPR-based Programmable Repression System for Neisseria gonorrhoeae

Description

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) are prokaryotic adaptive immune systems regularly utilized as DNA editing tools. While Neisseria gonorrhoeae does not have an endogenous CRISPR, the commensal species Neisseria lactamica encodes a functional Type I-C CRISPR-Cas system. We have established an IPTG-inducible, CRISPR-interference (CRISPRi) platform based on the N. lactamica Type I-C CRISPR missing the Cas3 nuclease to allow locus-specific transcriptional repression. As proof of principle, we targeted a non-phase-variable version of the opaD gene. We show that CRISPRi can downregulate opaD gene and protein expression, resulting in bacterial inability to stimulate neutrophil oxidative responses and to bind to an N-terminal fragment of CEACAM1. Importantly, we used CRISPRi to effectively knockdown all the transcripts of all eleven opa genes using a five-spacer CRISPR array, allowing control of the entire phase-variable opa family in strain FA1090. We also report that repression is reversible following IPTG-removal. Finally, we showed that the Type I-C CRISPRi system can conditionally reduce the expression of two essential genes. This CRISPRi system will allow the interrogation of every Gc gene, essential and non-essential, to study physiology and pathogenesis, and aid in antimicrobial development.

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