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A truncated anti-CRISPR protein prevents spacer acquisition but not interference.

ABSTRACT: CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference activity. Here, we show that S. thermophilus can block ACR-containing phages when the CRISPR immunity specifically targets the acr gene. This in turn selects for phage mutants carrying a deletion within the acr gene. Remarkably, a truncated acrIIA allele, found in a wild-type virulent streptococcal phage, does not block the interference activity of Cas9 but still prevents the acquisition of new immunities, thereby providing an example of an ACR specifically inhibiting spacer acquisition.

SUBMITTER: Philippe C

PROVIDER: S-EPMC9120153 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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A truncated anti-CRISPR protein prevents spacer acquisition but not interference.

Philippe Cécile C Morency Carlee C Plante Pier-Luc PL Zufferey Edwige E Achigar Rodrigo R Tremblay Denise M DM Rousseau Geneviève M GM Goulet Adeline A Moineau Sylvain S

Nature communications 20220519 1

CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference a ...[more]

PMID: 35589712

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Project description:BackgroundCRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.ResultsWe describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner.ConclusionsThe E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster--in our case the first acquired spacer--and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.

Dataset Information

A truncated anti-CRISPR protein prevents spacer acquisition but not interference.

Publications

A truncated anti-CRISPR protein prevents spacer acquisition but not interference.

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