A roadmap for gene functional characterisation in crops with large genomes: Lessons from polyploid wheat

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Abstract

Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite their importance, a lack of genomic information and resources has hindered the functional characterisation of genes in major crops. The recent release of high-quality reference sequences for these crops underpins a suite of genetic and genomic resources that support basic research and breeding. For wheat, these include gene model annotations, expression atlases and gene networks that provide information about putative function. Sequenced mutant populations, improved transformation protocols and structured natural populations provide rapid methods to study gene function directly. We highlight a case study exemplifying how to integrate these resources. This review provides a helpful guide for plant scientists, especially those expanding into crop research, to capitalise on the discoveries made in Arabidopsis and other plants. This will accelerate the improvement of crops of vital importance for food and nutrition security.

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Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

Zhen Liang, Kunling Chen, Tingdong Li … (2017)

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

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Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Yi Zhang, Zhen Liang, Yuan Zong … (2016)

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

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Genome editing in rice and wheat using the CRISPR/Cas system.

Qiwei Shan, Yanpeng Wang, Jun. Li … (2014)

Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1-2 weeks in protoplasts, and mutated rice plants can be generated within 13-17 weeks.

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Author and article information

Contributors

Cristobal Uauy:

ORCID: https://orcid.org/0000-0002-9814-1770

Christian S Hardtke: Role: Senior Editor

Christian S Hardtke: Role: Reviewing Editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 24 March 2020

Publication date Collection: 2020

Volume: 9

Electronic Location Identifier: e55646

Affiliations

[1 ]John Innes Centre, Norwich Research Park NorwichUnited Kingdom

[2 ]School of Biosciences, University of Birmingham BirminghamUnited Kingdom

[3 ]John Bingham Laboratory CambridgeUnited Kingdom

[4 ]Commonwealth Scientific and Industrial Research Organisation, Agriculture and Food (CSIRO) CanberraAustralia

[5 ]Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics Fiorenzuola d'ArdaItaly

[6 ]European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus HinxtonUnited Kingdom

[7 ]Rothamsted Research HarpendenUnited Kingdom

[8 ]Queensland Alliance for Agriculture and Food Innovation, The University of Queensland St LuciaAustralia

[9 ]Division of Plant and Crop Sciences, The University of Nottingham, Sutton Bonington Campus LoughboroughUnited Kingdom

[10 ]Department of Agricultural and Food Sciences (DISTAL), Alma Mater Studiorum - Università di Bologna (University of Bologna) BolognaItaly

[11 ]Crop Development Centre, University of Saskatchewan SaskatoonCanada

[12 ]International Maize and Wheat Improvement Center (CIMMYT) El BatánMexico

University of Lausanne Switzerland

Author notes

[†]

These authors contributed equally to this work.

Author information

Nikolai M Adamski https://orcid.org/0000-0003-1329-5138

Philippa Borrill https://orcid.org/0000-0002-7623-8256

Jemima Brinton https://orcid.org/0000-0001-7027-0450

Sophie A Harrington https://orcid.org/0000-0003-0754-0678

Clémence Marchal https://orcid.org/0000-0003-1669-3270

Luigi Cattivelli http://orcid.org/0000-0002-6067-4600

Wendy Harwood http://orcid.org/0000-0002-4323-9009

Sadiye Hayta http://orcid.org/0000-0003-2920-4720

Kostya Kanyuka http://orcid.org/0000-0001-6324-4123

Ricardo H Ramirez-Gonzalez http://orcid.org/0000-0001-5745-7085

Carolina Sansaloni http://orcid.org/0000-0003-2675-4524

Luzie U Wingen http://orcid.org/0000-0002-4682-4764

Brande BH Wulff https://orcid.org/0000-0003-4044-4346

Cristobal Uauy https://orcid.org/0000-0002-9814-1770

Article

Publisher ID: 55646

DOI: 10.7554/eLife.55646

PMC ID: 7093151

PubMed ID: 32208137

SO-VID: 22094d85-0143-445b-81d5-32aa6f3a9319

License:

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.