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

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) 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 isopropyl β-d-1-thiogalactopyranoside added (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 11 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. IMPORTANCE Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have proven instrumental in genetically manipulating many eukaryotic and prokaryotic organisms. Despite its usefulness, a CRISPR system had yet to be developed for use in Neisseria gonorrhoeae (Gc), a bacterium that is the main etiological agent of gonorrhea infection. Here, we developed a programmable and IPTG-inducible Type I-C CRISPR interference (CRISPRi) system derived from the commensal species Neisseria lactamica as a gene repression system in Gc. As opposed to generating genetic knockouts, the Type I-C CRISPRi system allows us to block transcription of specific genes without generating deletions in the DNA. We explored the properties of this system and found that a minimal spacer array is sufficient for gene repression while also facilitating efficient spacer reprogramming. Importantly, we also show that we can use CRISPRi to knockdown genes that are essential to Gc that cannot normally be knocked out under laboratory settings. Gc encodes ~800 essential genes, many of which have no predicted function. We predict that this Type I-C CRISPRi system can be used to help categorize gene functions and perhaps contribute to the development of novel therapeutics for gonorrhea.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation

“…Loss of Lst was confirmed by PCR, and the inability to sialylate itself was verified by 6B4 exclusion (see below). OpaD expression was confirmed by Western blotting bacterial lysates with 4B12 mAb against Opa ( 88 , 89 ).…”

A Toolbox forNeisseria meningitidis: Gene Editing, Complementation and Labelling

“…Finally, Geslewitz et al . (Geslewitz et al, 2023) proposed very recently to use a CRISPR interference strategy to inhibit the expression of essential genes in N. gonorrhoeae. Besides markerless mutation, there are few genetic complementation tools and they have all been developed for N. gonorrhoeae .…”

“…In addition, all three strategies are subject to spontaneous mutation of their selective marker, an event that is likely to generate false-positive mutants and which limits the use of these tools in models of bacteria with a high mutation rate, as in the case of strain Z5463, one of the model organisms of N. meningitidis (Achtman et al, 1988). Finally, Geslewitz et al (Geslewitz et al, 2023) proposed very recently to use a CRISPR interference strategy to inhibit the expression of essential genes in N. gonorrhoeae.…”