How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration
Abstract
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 spacermore »
- Authors:
-
- Cornell Univ., Ithaca, NY (United States)
- Pohang Univ. of Science and Technology (South Korea)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE; NIH/NIGMS; National Science Foundation (NSF)
- OSTI Identifier:
- 1430354
- Grant/Contract Number:
- AC02-06CH11357; GM118174; GM102543; P41-GM103403; S10-RR029205; DMR-1332208; GM-103485
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature (London)
- Additional Journal Information:
- Journal Name: Nature (London); Journal Volume: 550; Journal Issue: 7674; Journal ID: ISSN 0028-0836
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; DNA-binding proteins; Enzyme mechanisms; X-ray crystallography
Citation Formats
Xiao, Yibei, Ng, Sherwin, Nam, Ki Hyun, and Ke, Ailong. How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration. United States: N. p., 2017.
Web. doi:10.1038/nature24020.
Xiao, Yibei, Ng, Sherwin, Nam, Ki Hyun, & Ke, Ailong. How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration. United States. https://doi.org/10.1038/nature24020
Xiao, Yibei, Ng, Sherwin, Nam, Ki Hyun, and Ke, Ailong. Mon .
"How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration". United States. https://doi.org/10.1038/nature24020. https://www.osti.gov/servlets/purl/1430354.
@article{osti_1430354,
title = {How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration},
author = {Xiao, Yibei and Ng, Sherwin and Nam, Ki Hyun and Ke, Ailong},
abstractNote = {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. Here, we derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.},
doi = {10.1038/nature24020},
journal = {Nature (London)},
number = 7674,
volume = 550,
place = {United States},
year = {Mon Sep 04 00:00:00 EDT 2017},
month = {Mon Sep 04 00:00:00 EDT 2017}
}
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