Abstract
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.
Highlights
Prokaryotes have evolved an adaptive immune system called CRISPR/Cas that enables them to counter invasions from viruses and plasmids
CRISPR transcripts are cleaved into mature CRISPR RNAs and these remain bound by the ribonucleoprotein complex Cascade (Cascomplex for antiviral defence, in Type I-E consisting of proteins encoded by cas genes cse1, cse2, cas7, cas5 and cas6e) to guide the interference machinery to target DNA sequences [12]
New spacers are selected in a non-random process that takes into account the presence of a protospacer adjacent motif (PAM) on the target DNA
Summary
Prokaryotes have evolved an adaptive immune system called CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated protein) that enables them to counter invasions from viruses and plasmids (reviewed by [1,2,3,4]). This immune system contains genomic CRISPR loci in which genetic material from invaders is incorporated. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is likely degraded by Cas during CRISPR interference
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