Dataset Information

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.

ABSTRACT: Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

SUBMITTER: Plateau P

PROVIDER: S-EPMC6643042 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.

Plateau Pierre P Moch Clara C Blanquet Sylvain S

The Journal of biological chemistry 20190606 29

Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal C ...[more]

PMID: 31171718

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Project description:CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.

Dataset Information

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase.

Publications

Spermidine strongly increases the fidelity of <i>Escherichia coli</i> CRISPR Cas1-Cas2 integrase.

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