Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria

Govindarajan, Sutharsan; Borges, Adair L.; Karambelkar, Shweta; Bondy‐Denomy, Joseph

doi:10.1128/jb.00105-22

Cited by 9 publications

(5 citation statements)

References 57 publications

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“…This result contrasts with the expression of the Type II-nuclease dead Cas9 CRISPRi systems in different bacteria, where the expression must be fine tuned to prevent toxicity, even when using a non-targeting gRNA ( 77 – 79 ). While the expression of foreign proteins alone may be impacting bacterial fitness, this result does not exclude the possibility that the Cascade proteins are still non-specifically associated with the DNA during the search for a PAM and target sequence, as is seen by different Type I CRISPR systems in other bacteria ( 80 , 81 ).…”

Section: Discussionmentioning

confidence: 95%

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

Geslewitz,

Cardenas,

Zhou

et al. 2024

mBio

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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.

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Section: Discussionmentioning

confidence: 95%

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

Geslewitz,

Cardenas,

Zhou

et al. 2024

mBio

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“…S2). The coexistence of the protospacer with the cognate spacer has been proposed as representing autoimmunity processes with a negative effect on the bacterium ( 37 ), although this may also be explained by the fact that the prophage is expressing anti-CRISPR systems ( 38 ). However, other studies have shown that CRISPR-Cas systems can prevent the lytic cycle of phages but tolerate the virus integration as a prophage, allowing the bacteria to co-opt the phage genes for possible use as virulence factors ( 39 ).…”

Section: Discussionmentioning

confidence: 99%

Analysis of bacterial pangenomes reduces CRISPR dark matter and reveals strong association between membranome and CRISPR-Cas systems

et al. 2023

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CRISPR-Cas systems are prokaryotic acquired immunity mechanisms, which are found in 40% of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. However, the vast majority of these spacers have not yet been associated with the virus they recognize, and it has been named CRISPR dark matter. By analyzing the spacers of tens of thousands of genomes from six bacterial species, we have been able to reduce the CRISPR dark matter from 80% to as low as 15% in some of the species. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular sets of membrane proteins. Our results suggest that when bacteria present membrane proteins that make it compete better in its environment and these proteins are, in turn, receptors for specific phages, they would be forced to acquire CRISPR-Cas.

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“…S1). The coexistence of the protospacer with the cognate spacer has been proposed as representing autoimmunity processes with a negative effect on the bacterium (61), although this may also be explained by the fact that the prophage is expressing anti-CRISPR systems (62). However, other studies have shown that CRISPR-Cas systems can prevent the lytic cycle of phages but tolerate the virus integration as a prophage, allowing the bacteria to co-opt the phage genes for possible use as virulence factors (63).…”

Section: Discussionmentioning

confidence: 99%

Reducing CRISPR dark matter reveals a strong association between the bacterial membranome and CRISPR-Cas systems

Rubio

Sprang

Garzón

et al. 2022

Preprint

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Antimicrobial resistance is widely recognized as a serious global public health problem. To combat this threat, a thorough understanding of bacterial genomes is necessary. The current wide availability of bacterial genomes provides us with an in-depth understanding of the great variability of dispensable genes and their relationship with antimicrobials. Some of these accessory genes are those involved in CRISPR-Cas systems, which are acquired immunity systems that are present in part of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. But the vast majority of these spacers have not yet been associated with the virus they recognize, and this has been named CRISPR dark matter (spacers without associated recognized sequence). By analyzing the spacers of tens of thousands of genomes from six bacterial species highly resistant to antibiotics, we have been able to reduce the CRISPR dark matter from 80-90% to 15% in some cases. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular collections of membrane proteins. Our results suggest that when a bacterium presents membrane proteins that make it compete better in its environment, and these proteins are in turn receptors for specific phages, it would be forced to acquire CRISPR-Cas immunity systems to avoid infection by these phages.

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Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria

Abstract: CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood.

Cited by 9 publications

References 57 publications

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

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

Analysis of bacterial pangenomes reduces CRISPR dark matter and reveals strong association between membranome and CRISPR-Cas systems

Reducing CRISPR dark matter reveals a strong association between the bacterial membranome and CRISPR-Cas systems

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