Journal ArticleDOI
CRISPR-Cas9-mediated genome editing in apple and grapevine.
Yuriko Osakabe,Zhenchang Liang,Chong Ren,Chikako Nishitani,Keishi Osakabe,Masato Wada,Sadao Komori,Mickael Malnoy,Riccardo Velasco,Michele Poli,Minhee Jung,Ok Jae Koo,Roberto Viola,Chidananda Nagamangala Kanchiswamy +13 more
TLDR
This protocol provides a stepwise protocol for the design and transfer of CRISPR–Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components.Abstract:
The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR-Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR-Cas9 RNPs takes as little as 2-3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.read more
Citations
More filters
Journal ArticleDOI
Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing.
TL;DR: The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed and examples in the development of stimuli-responsive and targeted carriers for R NP delivery are highlighted.
Journal ArticleDOI
Genome Editing in Plants: Exploration of Technological Advancements and Challenges
Sanskriti Vats,Surbhi Kumawat,Virender Kumar,Gunvant Patil,Trupti Joshi,Humira Sonah,Tilak Raj Sharma,Rupesh Deshmukh +7 more
TL;DR: In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs, and a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis is provided.
Journal ArticleDOI
Genome editing for horticultural crop improvement.
Jiemeng Xu,Kai Hua,Zhaobo Lang +2 more
TL;DR: The combination of rapidly advancing genome-editing technology with breeding will greatly increase horticultural crop production and quality.
Journal ArticleDOI
Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique
Lígia Erpen-Dalla Corte,Lamiaa M. Mahmoud,Lamiaa M. Mahmoud,Tatiana S. Moraes,Zhonglin Mou,Jude W. Grosser,Manjul Dutt +6 more
TL;DR: Various aspects of the CRISPR/Cas9 system are explored, including a general presentation of the function of theCRISPR-associated protein 9 system in bacteria and its practical application as a biotechnological tool for editing plant genomes, particularly in horticultural crops.
Journal ArticleDOI
Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters.
TL;DR: In this paper, the authors used the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugarrelated tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%.
References
More filters
Journal ArticleDOI
The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.
Olivier Jaillon,Jean-Marc Aury,Benjamin Noel,Alberto Policriti,Christian Clepet,Alberto Casagrande,Nathalie Choisne,Sébastien Aubourg,Nicola Vitulo,Claire Jubin,Alessandro Vezzi,Fabrice Legeai,Philippe Hugueney,Corinne Dasilva,David S. Horner,Erica Mica,Delphine Jublot,Julie Poulain,Clémence Bruyère,Alain Billault,Béatrice Segurens,Michel Gouyvenoux,Edgardo Ugarte,Federica Cattonaro,Véronique Anthouard,Virginie Vico,Cristian Del Fabbro,Michael Alaux,Gabriele Di Gaspero,Vincent Dumas,Nicoletta Felice,Sophie Paillard,Irena Juman,Marco Moroldo,Simone Scalabrin,Aurélie Canaguier,Isabelle Le Clainche,G Malacrida,Eléonore Durand,Graziano Pesole,Valérie Laucou,Philippe Chatelet,Didier Merdinoglu,Massimo Delledonne,Mario Pezzotti,Alain Lecharny,Claude Scarpelli,François Artiguenave,M. Enrico Pè,Giorgio Valle,Michele Morgante,Michel Caboche,Anne-Françoise Adam-Blondon,Jean Weissenbach,Francis Quetier,Patrick Wincker +55 more
TL;DR: A high-quality draft of the genome sequence of grapevine is obtained from a highly homozygous genotype, revealing the contribution of three ancestral genomes to the grapevine haploid content and explaining the chronology of previously described whole-genome duplication events in the evolution of flowering plants.
Journal ArticleDOI
A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.
K Edwards,C Johnstone,C Thompson +2 more
TL;DR: This work has developed a method for the rapid extraction of small amounts of plant genomic DNA suitable for PCR analysis that is applicable to a variety of plant species and has the added advantage of not requiring any phenol or chloroform extraction.
Journal ArticleDOI
Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
TL;DR: It is shown that complexes of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33% in human cells.
Journal ArticleDOI
The genome of the domesticated apple ( Malus × domestica Borkh.)
Riccardo Velasco,Andrey Zharkikh,Jason P. Affourtit,Amit Dhingra,Alessandro Cestaro,Ananth Kalyanaraman,Paolo Fontana,Satish Bhatnagar,Michela Troggio,Dmitry Pruss,Silvio Salvi,Massimo Pindo,Paolo Baldi,Sara Castelletti,Marina Cavaiuolo,G. Coppola,Fabrizio Costa,V. Cova,Antonio Dal Ri,Vadim V. Goremykin,M. Komjanc,Sara Longhi,P. Magnago,Giulia Malacarne,Mickael Malnoy,Diego Micheletti,Marco Moretto,Michele Perazzolli,Azeddine Si-Ammour,Silvia Vezzulli,E. Zini,Glenn Eldredge,Lisa M. Fitzgerald,N. Gutin,Jerry S. Lanchbury,Teresita Macalma,J.T. Mitchell,Julia Reid,Bryan Wardell,Chinnappa D. Kodira,Zhoutao Chen,Brian Desany,Faheem Niazi,Melinda Palmer,Tyson Koepke,Derick Jiwan,Scott Schaeffer,Vandhana Krishnan,Changjun Wu,Vu T. Chu,Stephen T. King,Jessica Vick,Quanzhou Tao,Amy Mraz,Aimee Stormo,Keith E. Stormo,Robert Bogden,Davide Ederle,Alessandra Stella,Alberto Vecchietti,Martin M. Kater,Simona Masiero,Pauline Lasserre,Yves Lespinasse,Andrew C. Allan,Vincent G. M. Bus,David Chagné,Ross N. Crowhurst,Andrew P. Gleave,Enrico Lavezzo,Jeffrey A. Fawcett,Jeffrey A. Fawcett,Sebastian Proost,Sebastian Proost,Pierre Rouzé,Pierre Rouzé,Lieven Sterck,Lieven Sterck,Stefano Toppo,Barbara Lazzari,Roger P. Hellens,Charles-Eric Durel,Alexander Gutin,Roger E. Bumgarner,Susan E. Gardiner,Mark H. Skolnick,Michael Egholm,Yves Van de Peer,Yves Van de Peer,Francesco Salamini,Roberto Viola +90 more
TL;DR: It is shown that a relatively recent (>50 million years ago) genome-wide duplication has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae, which partly support the monophyly of the ancestral paleohexaploidy of eudicots.
Journal ArticleDOI
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins
TL;DR: Delivery of purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells is delivered and RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off- target mutations associated with plasmid transfection at off-target sites.