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Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9

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Durr, Julius, Papareddy, Ranjith, Nakajima, Keiji and Gutierrez-Marcos, José F. (2018) Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9. Scientific Reports, 8 (1). 4443. doi:10.1038/s41598-018-22667-1 ISSN 2045-2322.

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Official URL: http://dx.doi.org/10.1038/s41598-018-22667-1

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Abstract

Genome editing using CRISPR/Cas9 is considered the best instrument for genome engineering in plants. This methodology is based on the nuclease activity of Cas9 that is guided to specific genome sequences by single guide RNAs (sgRNAs) thus enabling researchers to engineer simple mutations or large chromosomal deletions. Current methodologies for targeted genome editing in plants using CRISPR/Cas9 are however largely inefficient, mostly due to low Cas9 activity, variable sgRNA efficiency and low heritability of genetic lesions. Here, we describe a newly developed strategy to enhance CRISPR/Cas9 efficiency in Arabidopsis thaliana focusing on the design of novel binary vectors (pUbiCAS9-Red and pEciCAS9-Red), the selection of highly efficient sgRNAs, and the use of direct plant regeneration from induced cell cultures. Our work demonstrates that by combining these three independent developments, heritable targeted chromosomal deletions of large gene clusters and intergenic regulatory sequences can be engineered at a high efficiency. Our results demonstrate that this improved CRISPR/Cas9 methodology can provide a fast, efficient and cost-effective tool to engineer targeted heritable chromosomal deletions, which will be instrumental for future high-throughput functional genomics studies in plants.

Item Type: Journal Article
Subjects: Q Science > QK Botany
Divisions: Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH): Arabidopsis thaliana -- Genetic engineering, Plants -- Functional genomics
Journal or Publication Title: Scientific Reports
Publisher: Nature Publishing Group
ISSN: 2045-2322
Official Date: 13 March 2018
Dates:
DateEvent
13 March 2018Published
22 February 2018Accepted
Volume: 8
Number: 1
Article Number: 4443
DOI: 10.1038/s41598-018-22667-1
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Restricted or Subscription Access
Date of first compliant deposit: 15 March 2018
Date of first compliant Open Access: 15 March 2018
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
BB/L003023/1Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
BB/N005279/1 Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
BB/N00194X/1Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268

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