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Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR–Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helica...

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Autores principales: Musharova, Olga, Klimuk, Evgeny, Datsenko, Kirill A., Metlitskaya, Anastasia, Logacheva, Maria, Semenova, Ekaterina, Severinov, Konstantin, Savitskaya, Ekaterina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389516/
https://www.ncbi.nlm.nih.gov/pubmed/28204574
http://dx.doi.org/10.1093/nar/gkx097
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author Musharova, Olga
Klimuk, Evgeny
Datsenko, Kirill A.
Metlitskaya, Anastasia
Logacheva, Maria
Semenova, Ekaterina
Severinov, Konstantin
Savitskaya, Ekaterina
author_facet Musharova, Olga
Klimuk, Evgeny
Datsenko, Kirill A.
Metlitskaya, Anastasia
Logacheva, Maria
Semenova, Ekaterina
Severinov, Konstantin
Savitskaya, Ekaterina
author_sort Musharova, Olga
collection PubMed
description During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR–Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1–Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.
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spelling pubmed-53895162017-04-24 Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation Musharova, Olga Klimuk, Evgeny Datsenko, Kirill A. Metlitskaya, Anastasia Logacheva, Maria Semenova, Ekaterina Severinov, Konstantin Savitskaya, Ekaterina Nucleic Acids Res Molecular Biology During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR–Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1–Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array. Oxford University Press 2017-04-07 2017-02-15 /pmc/articles/PMC5389516/ /pubmed/28204574 http://dx.doi.org/10.1093/nar/gkx097 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Molecular Biology
Musharova, Olga
Klimuk, Evgeny
Datsenko, Kirill A.
Metlitskaya, Anastasia
Logacheva, Maria
Semenova, Ekaterina
Severinov, Konstantin
Savitskaya, Ekaterina
Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
title Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
title_full Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
title_fullStr Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
title_full_unstemmed Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
title_short Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
title_sort spacer-length dna intermediates are associated with cas1 in cells undergoing primed crispr adaptation
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389516/
https://www.ncbi.nlm.nih.gov/pubmed/28204574
http://dx.doi.org/10.1093/nar/gkx097
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