Dataset Information

CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.

ABSTRACT: Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.

SUBMITTER: Grainy J

PROVIDER: S-EPMC6698737 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.

Grainy Julie J Garrett Sandra S Graveley Brenton R BR P Terns Michael M

Nucleic acids research 20190801 14

Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat bord ...[more]

PMID: 31219587

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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 repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.

Publications

CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.

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