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CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system

CRISPR–Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of S...

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Autores principales: Kim, Jenny G, Garrett, Sandra, Wei, Yunzhou, Graveley, Brenton R, Terns, Michael P
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895254/
https://www.ncbi.nlm.nih.gov/pubmed/31392984
http://dx.doi.org/10.1093/nar/gkz677
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author Kim, Jenny G
Garrett, Sandra
Wei, Yunzhou
Graveley, Brenton R
Terns, Michael P
author_facet Kim, Jenny G
Garrett, Sandra
Wei, Yunzhou
Graveley, Brenton R
Terns, Michael P
author_sort Kim, Jenny G
collection PubMed
description CRISPR–Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3′ hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.
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spelling pubmed-68952542019-12-11 CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system Kim, Jenny G Garrett, Sandra Wei, Yunzhou Graveley, Brenton R Terns, Michael P Nucleic Acids Res Molecular Biology CRISPR–Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3′ hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity. Oxford University Press 2019-09-19 2019-08-08 /pmc/articles/PMC6895254/ /pubmed/31392984 http://dx.doi.org/10.1093/nar/gkz677 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ 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.
spellingShingle Molecular Biology
Kim, Jenny G
Garrett, Sandra
Wei, Yunzhou
Graveley, Brenton R
Terns, Michael P
CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system
title CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system
title_full CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system
title_fullStr CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system
title_full_unstemmed CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system
title_short CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system
title_sort crispr dna elements controlling site-specific spacer integration and proper repeat length by a type ii crispr–cas system
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895254/
https://www.ncbi.nlm.nih.gov/pubmed/31392984
http://dx.doi.org/10.1093/nar/gkz677
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