Dataset Information

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration.

ABSTRACT: CRISPR-Cas systems defend prokaryotes against viruses and plasmids. Short DNA segments of the invader, known as spacers, are stored in the CRISPR array as immunological memories. New spacers are added invariably to the 5' end of the array; therefore, the first spacer matches the latest foreign threat. Whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. Here we show that a conserved sequence located immediately upstream of the CRISPR array specifies the site of new spacer integration. Mutation of this sequence results in erroneous incorporation of new spacers into the middle of the array. We show that spacers added through polarized acquisition give rise to more robust CRISPR-Cas immunity than spacers added to the middle of the array. This study demonstrates that the CRISPR-Cas system specifies the site of spacer integration to optimize the immune response against the most immediate threat to the host.

SUBMITTER: McGinn J

PROVIDER: S-EPMC5096952 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration.

McGinn Jon J Marraffini Luciano A LA

Molecular cell 20160908 3

CRISPR-Cas systems defend prokaryotes against viruses and plasmids. Short DNA segments of the invader, known as spacers, are stored in the CRISPR array as immunological memories. New spacers are added invariably to the 5' end of the array; therefore, the first spacer matches the latest foreign threat. Whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. Here we show that a conserved sequence located immediately upstream of the CRISPR array ...[more]

PMID: 27618488

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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

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration.

Publications

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration.

Similar Datasets

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