Transcription termination and antitermination of bacterial CRISPR arrays

Stringer, Anne M.; Baniulyte, Gabriele; Lasek‐Nesselquist, Erica; Seed, Kimberley D.; Wade, Joseph T.

doi:10.7554/elife.58182

Cited by 11 publications

(9 citation statements)

References 69 publications

(84 reference statements)

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“…In serovar Typhimurium str. 14028s, 236 spacer- and Cascade-binding sites were identified using ChIP-seq of Cas5 40 . After mapping these sites on the complete genome of serovar Typhimurium str.…”

Section: Resultsmentioning

confidence: 99%

The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella

Kushwaha

Bhavesh

Abdella

et al. 2020

Sci Rep

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Salmonellae display intricate evolutionary patterns comprising over 2500 serovars having diverse pathogenic profiles. The acquisition and/or exchange of various virulence factors influences the evolutionary framework. To gain insights into evolution of Salmonella in association with the CRISPR-Cas genes we performed phylogenetic surveillance across strains of 22 Salmonella serovars. The strains differed in their CRISPR1-leader and cas operon features assorting into two main clades, CRISPR1-STY/cas-STY and CRISPR1-STM/cas-STM, comprising majorly typhoidal and non-typhoidal Salmonella serovars respectively. Serovars of these two clades displayed better relatedness, concerning CRISPR1-leader and cas operon, across genera than between themselves. This signifies the acquisition of CRISPR1/Cas region could be through a horizontal gene transfer event owing to the presence of mobile genetic elements flanking CRISPR1 array. Comparison of CRISPR and cas phenograms with that of multilocus sequence typing (MLST) suggests differential evolution of CRISPR/Cas system. As opposed to broad-host-range, the host-specific serovars harbor fewer spacers. Mapping of protospacer sources suggested a partial correlation of spacer content with habitat diversity of the serovars. Some serovars like serovar Enteritidis and Typhimurium that inhabit similar environment/infect similar hosts hardly shared their protospacer sources.

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Section: Resultsmentioning

confidence: 99%

The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella

Kushwaha

Bhavesh

Abdella

et al. 2020

Sci Rep

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“…boxA could improve RNA polymerase processivity of these and our synthetic arrays (Fig. 4 ) by a number of different mechanisms, one of which may be blocking Rho-dependent transcription termination 52 – 54 , as hypothesized by Stringer et al 37 . Still, as naturally occurring CRISPR arrays can stretch to the hundreds of spacers 32 , there could be additional unknown factors that regulate spacer transcription that could one day be applied to a CRISPR-based tool.…”

Section: Discussionmentioning

confidence: 60%

“…Recently Stringer et. al reported that boxA elements may be evolutionarily conserved upstream of naturally occurring CRISPR arrays where they appear to promote expression of downstream spacers within long arrays 37 .…”

Section: Resultsmentioning

confidence: 99%

“…Our data reiterate that this is an advantageous strategy as the earliest spacers in the array are the most likely to be transcribed, and therefore, will produce the greatest quantity of protective crRNAs for an ongoing viral attack. Several bacterial CRISPR systems appear to have overcome polarity of spacer expression through acquiring boxA sequences upstream of their arrays 37 . boxA could improve RNA polymerase processivity of these and our synthetic arrays (Fig.…”

Section: Discussionmentioning

confidence: 99%

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A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

et al. 2021

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Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

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“…Pre-crRNAs are amongst the longest non-coding RNAs in prokaryotes next to the 16S and 23S rRNAs. In E. coli , dedicated antitermination complexes ensure processivity of CRISPR array transcription by preventing premature Rho-dependent transcription termination similar to the antitermination complexes facilitating rRNA transcription [4]. Beyond E. coli , little is known about specific mechanisms controlling CRISPR array transcription.…”

Section: Introductionmentioning

confidence: 99%

Cbp1-Cren7 chromatinization of CRISPR arrays favours transcription from leader-over cryptic promoters

Blombach

Sýkora

Case

et al. 2023

Preprint

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CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbor extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposasons. Cbp1 recruits Cren7 forming together 'chimeric' chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third HTH domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

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Transcription termination and antitermination of bacterial CRISPR arrays

Cited by 11 publications

References 69 publications

The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella

The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

Cbp1-Cren7 chromatinization of CRISPR arrays favours transcription from leader-over cryptic promoters

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