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A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation

The Class 1 type I CRISPR–Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for funct...

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Autores principales: Xu, Zeling, Li, Yanran, Cao, Huiluo, Si, Meiru, Zhang, Guangming, Woo, Patrick C Y, Yan, Aixin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8450077/
https://www.ncbi.nlm.nih.gov/pubmed/34157103
http://dx.doi.org/10.1093/nar/gkab521
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author Xu, Zeling
Li, Yanran
Cao, Huiluo
Si, Meiru
Zhang, Guangming
Woo, Patrick C Y
Yan, Aixin
author_facet Xu, Zeling
Li, Yanran
Cao, Huiluo
Si, Meiru
Zhang, Guangming
Woo, Patrick C Y
Yan, Aixin
author_sort Xu, Zeling
collection PubMed
description The Class 1 type I CRISPR–Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λ(red) system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an ‘all-in-one’ I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in ‘non-model’ bacterial species.
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spelling pubmed-84500772021-09-20 A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation Xu, Zeling Li, Yanran Cao, Huiluo Si, Meiru Zhang, Guangming Woo, Patrick C Y Yan, Aixin Nucleic Acids Res Methods Online The Class 1 type I CRISPR–Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λ(red) system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an ‘all-in-one’ I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in ‘non-model’ bacterial species. Oxford University Press 2021-06-22 /pmc/articles/PMC8450077/ /pubmed/34157103 http://dx.doi.org/10.1093/nar/gkab521 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://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 Methods Online
Xu, Zeling
Li, Yanran
Cao, Huiluo
Si, Meiru
Zhang, Guangming
Woo, Patrick C Y
Yan, Aixin
A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
title A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
title_full A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
title_fullStr A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
title_full_unstemmed A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
title_short A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
title_sort transferrable and integrative type i-f cascade for heterologous genome editing and transcription modulation
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8450077/
https://www.ncbi.nlm.nih.gov/pubmed/34157103
http://dx.doi.org/10.1093/nar/gkab521
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