Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans

Benz, Fabienne; Camara-Wilpert, Sarah; Russel, Jakob; Wandera, Katharina G.; Čepaitė, Rimvydė; Ares-Arroyo, Manuel; Gomes-Filho, José Vicente; Englert, Frank; Kuehn, Johannes A.; Gloor, Silvana; Mestre, Mario Rodríguez; Cuénod, Aline; Aguilà-Sans, Mònica; Maccario, Lorrie; Egli, Adrian; Randau, Lennart; Pausch, Patrick; Rocha, Eduardo P.C.; Beisel, Chase L.; Madsen, Jonas Stenløkke; Bikard, David; Hall, Alex R.; Sørensen, Søren Johannes; Pinilla-Redondo, Rafael

doi:10.1016/j.chom.2024.04.016

Cited by 3 publications

(5 citation statements)

References 107 publications

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“…We also showed that CasDinG is not required for Type IV-A1 activity if its target is a promoter sequence. Consistent with recently reported similar observations 12 , this observation indicates that the crRNP is able to inhibit transcription initiation. Together, our findings suggest a transcription-dependent role of CasDinG in modulating Type IV-A CRISPR-Cas activity.…”

Section: Discussionsupporting

confidence: 93%

“…Our observations support previous findings showing that (i) the Type IV-A1 system of P. oleovorans reduces transcript levels in an extended area surrounding its native chromosomal target site 9 and that (ii) CasDinG is dispensable for targeting promoter regions 12 . The extended CasDinG-mediated regional effects of Type IV-A1 CRISPRi has implications for applications in CRISPR-Cas-mediated gene regulation that complement dCas9-mediated CRISPRi methodology 17,18 .…”

Section: Influence Of Synthetic Crrnas On Sfgfp Gene Expression In P ...supporting

confidence: 92%

“…In addition, a CRISPR knockout (ΔCRISPR) strain was generated which does not contain competing natural crRNAs of the wild-type strain, including the self-targeting crRNA that represses expression of the pilN gene 9 . As CasDinG was observed to be essential for Type IV-A CRISPR-Cas activity 12,15 , we also created a CasDinG knockout (Δ dinG ) strain. Both deletion strains expressed sfGFP from an arabinose inducible promoter.…”

Section: Resultsmentioning

confidence: 99%

“…1a). CasDinG was identified to be essential for plasmid targeting and gene silencing, as mutations that affect its helicase and ATPase activities abolished CRISPR-Cas interference 9,10,[12][13][14] . The protein's helicase domain exhibits ATP-dependent 5′-3′ DNA translocase activity, enabling the unwinding of DNA and RNA/DNA duplexes 15 .…”

mentioning

confidence: 99%

“…The protein's helicase domain exhibits ATP-dependent 5′-3′ DNA translocase activity, enabling the unwinding of DNA and RNA/DNA duplexes 15 . However, CasDinG was found to be dispensable for gene repression when the effector complex was targeting the promoter region of a gene 12 .…”

mentioning

confidence: 99%

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Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

Sanchez-Londono,

Rust,

Hernández-Tamayo

et al. 2024

Preprint

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Type IV CRISPR-Cas effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system ofPseudomonas oleovoransadditionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference inP. oleovoransandEscherichia colicells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA-Seq analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range down-regulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.

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Section: Discussionsupporting

confidence: 93%

Section: Influence Of Synthetic Crrnas On Sfgfp Gene Expression In P ...supporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

Sanchez-Londono,

Rust,

Hernández-Tamayo

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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Going viral: The role of mobile genetic elements in bacterial immunity

Beamud,

Benz,

Bikard

2024

Cell Host & Microbe

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Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

Sanchez-Londono,

Rust,

Hernández-Tamayo

et al. 2024

Nucleic Acids Research

View full text Add to dashboard Cite

Type IV CRISPR–Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR–Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA sequencing (RNA-seq) analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range downregulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.

show abstract

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans

Cited by 3 publications

References 107 publications

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

Going viral: The role of mobile genetic elements in bacterial immunity

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

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