scispace - formally typeset
Search or ask a question
Book ChapterDOI

Genome Editing in Apple

TL;DR: This chapter will provide an overview of current studies of genome editing in apple and will discern and explore how this strategy will provide insights into molecular breeding technologies for genetic improvement of the apple.
Abstract: Genome editing with artificially engineered nucleases is an advanced molecular technology for pursuing precise and effective genetic engineering. In this technology, engineered nucleases induce DNA double-strand breaks at targeted sites in a genome, stimulating the DNA repair system in cells, thus enabling site-directed mutagenesis. Genome editing using CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein9 (Cas9), originating from a defence system of prokaryotes, is a powerful technology that is now being widely utilized in molecular research studies, as well as in breeding programmes of various plant species, including fruit trees, to impart either novel or enhanced traits to established commercial cultivars or to new cultivars/genotypes. Recently, several reports have demonstrated successful apple genome editing and the introduction of important traits, such as those for early flowering and reduced fire blight susceptibility, to popular commercial cultivars, such as ‘Gala’ and ‘Golden Delicious’. It is important to point out that these reports reveal that such genome-edited/mutant apple plants or cell lines do not carry foreign genes. Nevertheless, during the process of precise genome editing, the coexistence of various types of mutations referred to as “mosaic mutations” and off-target effects are major concerns. Therefore, to minimize such effects, selection of target sequences and estimation of off-target effects for CRISPR/Cas9 has been developed for many organisms, and these have also been employed for apple by using in silico analysis based on genome information. On the other hand, apple genome heterozygosity has led to difficulties in genome editing, as the complex genome of apple precludes the use of some of these basic techniques for genome editing. Therefore, further studies focused on genome information and culture techniques tailored for apple are needed. It will be highly critical for each apple cultivar in developing precise and efficient genome editing for apple. This chapter will provide an overview of current studies of genome editing in apple and will discern and explore how this strategy will provide insights into molecular breeding technologies for genetic improvement of the apple.
Citations
More filters
Journal ArticleDOI
TL;DR: This paper reviewed the most relevant mutations for breeding traits in Prunus species such as flowering time, self-compatibility, fruit quality, and disease tolerance, including new molecular perspectives in the present postgenomic era including CRISPR/Cas9 and TALEN editing technologies.
Abstract: Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the Prunus species, as well as in the rest of crop species. Mutation can occur in nature at a very low frequency or can be induced artificially. Spontaneous or bud sport mutations in somatic cells can be vegetatively propagated to get an individual with the mutant phenotype. Unlike animals, plants have unlimited growth and totipotent cells that let somatic mutations to be transmitted to the progeny. On the other hand, in vitro tissue culture makes it possible to induce mutation in plant material and perform large screenings for mutant’s selection and cleaning of chimeras. Finally, targeted mutagenesis has been boosted by the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 and Transcription activator-like effector nuclease (TALEN) editing technologies. Over the last few decades, environmental stressors such as global warming have been threatening the supply of global demand for food based on population growth in the near future. For this purpose, the release of new varieties adapted to such changes is a requisite, and selected or generated Prunus mutants by properly regulated mechanisms could be helpful to this task. In this work, we reviewed the most relevant mutations for breeding traits in Prunus species such as flowering time, self-compatibility, fruit quality, and disease tolerance, including new molecular perspectives in the present postgenomic era including CRISPR/Cas9 and TALEN editing technologies.

3 citations

References
More filters
Journal ArticleDOI
TL;DR: It is reported that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies.
Abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

1,878 citations

Journal ArticleDOI
Riccardo Velasco, Andrey Zharkikh1, Jason P. Affourtit2, Amit Dhingra3, Alessandro Cestaro, Ananth Kalyanaraman3, Paolo Fontana, Satish Bhatnagar1, Michela Troggio, Dmitry Pruss1, Silvio Salvi4, 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 Eldredge1, Lisa M. Fitzgerald1, N. Gutin1, Jerry S. Lanchbury1, Teresita Macalma1, J.T. Mitchell1, Julia Reid1, Bryan Wardell1, Chinnappa D. Kodira2, Zhoutao Chen2, Brian Desany2, Faheem Niazi2, Melinda Palmer2, Tyson Koepke3, Derick Jiwan3, Scott Schaeffer3, Vandhana Krishnan3, Changjun Wu3, Vu T. Chu5, Stephen T. King5, Jessica Vick5, Quanzhou Tao, Amy Mraz, Aimee Stormo, Keith E. Stormo, Robert Bogden, Davide Ederle6, Alessandra Stella6, Alberto Vecchietti6, Martin M. Kater7, Simona Masiero7, Pauline Lasserre, Yves Lespinasse, Andrew C. Allan8, Vincent G. M. Bus8, David Chagné8, Ross N. Crowhurst8, Andrew P. Gleave8, Enrico Lavezzo9, Jeffrey A. Fawcett10, Jeffrey A. Fawcett11, Sebastian Proost11, Sebastian Proost10, Pierre Rouzé10, Pierre Rouzé11, Lieven Sterck10, Lieven Sterck11, Stefano Toppo9, Barbara Lazzari6, Roger P. Hellens8, Charles-Eric Durel, Alexander Gutin1, Roger E. Bumgarner5, Susan E. Gardiner8, Mark H. Skolnick1, Michael Egholm2, Yves Van de Peer11, Yves Van de Peer10, Francesco Salamini6, Roberto Viola 
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.
Abstract: We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

1,718 citations

Journal ArticleDOI
TL;DR: It is found that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro, and it is demonstrated that the best predictions can significantly reduce the time spent on guide screening.
Abstract: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

1,256 citations

Journal ArticleDOI
TL;DR: The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (Cas9) system is described, a recently developed tool for the introduction of site-specific double-stranded DNA breaks and the strengths and weaknesses are highlighted.

948 citations

Journal ArticleDOI
TL;DR: Transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%.
Abstract: Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

900 citations