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Molecular recordings by directed CRISPR spacer acquisition.

ABSTRACT: The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

SUBMITTER: Shipman SL

PROVIDER: S-EPMC4994893 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Molecular recordings by directed CRISPR spacer acquisition.

Shipman Seth L SL Nivala Jeff J Macklis Jeffrey D JD Church George M GM

Science (New York, N.Y.) 20160609 6298

The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into ...[more]

PMID: 27284167

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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

Molecular recordings by directed CRISPR spacer acquisition.

Publications

Molecular recordings by directed CRISPR spacer acquisition.

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