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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
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WRAP-highly-efficient-heritable-targeted-deletions-gene-clusters-non-coding-regulatory-regions-Arabidopsis-Gutierrez-Marcos-2018.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (5Mb) | Preview |
Official URL: http://dx.doi.org/10.1038/s41598-018-22667-1
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 | ||||||||||||
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| Subjects: | Q Science > QK Botany | ||||||||||||
| Divisions: | Faculty of 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: |
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| Date of first compliant deposit: | 15 March 2018 | ||||||||||||
| 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 | ||||||||||||
| RIOXX Funder/Project Grant: |
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