Prespacer processing and specific integration in a Type I-A CRISPR system

Clare Rollie; Shirley Graham; Christophe Rouillon; Malcolm F. White

doi:10.1093/nar/gkx1232

Prespacer processing and specific integration in a Type I-A CRISPR system

Clare Rollie, Shirley Graham, Christophe Rouillon, Malcolm F. White^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types.

Original language	English
Pages (from-to)	1007-1020
Number of pages	14
Journal	Nucleic Acids Research
Volume	46
Issue number	3
Early online date	8 Dec 2017
DOIs	https://doi.org/10.1093/nar/gkx1232
Publication status	Published - 16 Feb 2018

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10.1093/nar/gkx1232Licence: CC BY

Rollie-2017-Prespacer-processing-NAR-gkx1232-CC
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org / licenses / by / 4.0 / ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Final published version, 3.74 MBLicence: CC BY

CRISPR Adaption: CRISPR Adaptation- the basis for prokaryotic adaptive immunity.
White, M. (PI)
BBSRC
1/10/15 → 31/12/18
Project: Standard

Malcolm White
- mfw2st-andrews.acuk
- School of Biology - Professor
- Biomedical Sciences Research Complex
Person: Academic

Cite this

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title = "Prespacer processing and specific integration in a Type I-A CRISPR system",

abstract = "The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types.",

author = "Clare Rollie and Shirley Graham and Christophe Rouillon and White, {Malcolm F.}",

note = "This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (REF: BB/M021017/1 to MFW).",

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AU - Rouillon, Christophe

AU - White, Malcolm F.

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Prespacer processing and specific integration in a Type I-A CRISPR system

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CRISPR Adaption: CRISPR Adaptation- the basis for prokaryotic adaptive immunity.

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Malcolm White

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