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Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss

CRISPR–Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation....

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Autores principales: Rao, Chitong, Chin, Denny, Ensminger, Alexander W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602111/
https://www.ncbi.nlm.nih.gov/pubmed/28724535
http://dx.doi.org/10.1261/rna.062083.117
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author Rao, Chitong
Chin, Denny
Ensminger, Alexander W.
author_facet Rao, Chitong
Chin, Denny
Ensminger, Alexander W.
author_sort Rao, Chitong
collection PubMed
description CRISPR–Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR–Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3′–5′ translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.
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spelling pubmed-56021112018-10-01 Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss Rao, Chitong Chin, Denny Ensminger, Alexander W. RNA Article CRISPR–Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR–Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3′–5′ translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation. Cold Spring Harbor Laboratory Press 2017-10 /pmc/articles/PMC5602111/ /pubmed/28724535 http://dx.doi.org/10.1261/rna.062083.117 Text en © 2017 Rao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Article
Rao, Chitong
Chin, Denny
Ensminger, Alexander W.
Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss
title Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss
title_full Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss
title_fullStr Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss
title_full_unstemmed Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss
title_short Priming in a permissive type I-C CRISPR–Cas system reveals distinct dynamics of spacer acquisition and loss
title_sort priming in a permissive type i-c crispr–cas system reveals distinct dynamics of spacer acquisition and loss
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602111/
https://www.ncbi.nlm.nih.gov/pubmed/28724535
http://dx.doi.org/10.1261/rna.062083.117
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