CRISPR/Cas9-mediated targeted mutagenesis in grape

PLOS ONE, Dec 2019

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

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CRISPR/Cas9-mediated targeted mutagenesis in grape

May CRISPR/Cas9-mediated targeted mutagenesis in grape Ikuko Nakajima 0 1 2 Yusuke Ban 0 1 2 Akifumi Azuma 0 1 2 Noriyuki Onoue 0 1 2 Takaya Moriguchi 0 1 2 Toshiya Yamamoto 0 1 2 Seiichi Toki 0 2 Masaki Endo 0 2 0 Current address: Western Region Agricultural Research Center, National Agriculture and Food Research Organization , Nishifukatsu-cho, Fukuyama, Hiroshima , Japan 1 Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization , Fujimoto, Tsukuba, Ibaraki , Japan , 2 Institute of Agrobiological Sciences, National Agriculture and Food Research Organization , Kannondai, Tsukuba, Ibaraki , Japan , 3 Graduate School of Nanobioscience, Yokohama City University , Seto, Kanazawa-ku, Yokohama, Kanagawa , Japan , 4 Kihara Institute for Biological Research, Yokohama City University , Maioka-cho, Yokohama, Kanagawa , Japan 2 Editor: Sara Amancio, Universidade de Lisboa Instituto Superior de Agronomia , PORTUGAL RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grapeÐan important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Cabinet Office, Government of Japan, Cross-Ministerial Strategic Innovation Promotion Program (SIP), "Technologies for creating next-generation agriculture, forestry and fisheries" (funding agency: Bio-oriented Technology Research Advancement Institution, NARO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Functional analysis of any gene of interest in plant genomes has traditionally relied heavily on the use of transfer DNA (T-DNA) and transposon insertional mutagenesis, or chemical- and irradiation-induced mutagenesis, to generate mutants. However, saturation mutagenesis is difficult in non-model plants due to a lack of genome information, large genome size and/or low transformation efficiency. Recent progress in the use of sequence-specific nucleases (SSNs) has opened new opportunities for reverse genetics in plants. SSNs, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (Cas9) system, have been developed to induce DNA double-strand breaks (DBSs) at specific genome sites. DSBs have a high propensity to induce site-directed mutations through errorprone genome repair via non-homologous end joining (NHEJ). Once transgenic cells expressing SSN constructs are obtained, various mutations will be induced at the specific target locus in independent cells, meaning that the limitations of low transformation efficiency can be overcome by the high performance of SSNs. Of the SSNs now available, the CRISPR/Cas9 system has been utilized successfully for mutagenesis in a variety of organisms, including plants such as Arabidopsis, sorghum, rice, tomato, maize, wheat, potato, poplar, orange, liverwort, petunia, and cucumber (for review, see Bortesi and Fischer [ 1 ]). In addition, very recently, successful CRISPR/Cas9 mediated targeted mutagenesis in grape was reported by Ren et al. [ 2 ]. Unlike ZFNs or TALENs, the CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple single guide RNAs (sgRNAs) for each target gene. Furthermore, only 20 nt in the sgRNA sequence need to be changed to confer a different target specificity, which means that complicated cloning is also unnecessary. This allows the inexpensive assembly of large sgRNA libraries so that the CRISPR/Cas9 system can be used for highthroughput functional genomics applications, bringing genome editing within the budget (...truncated)


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Ikuko Nakajima, Yusuke Ban, Akifumi Azuma, Noriyuki Onoue, Takaya Moriguchi, Toshiya Yamamoto, Seiichi Toki, Masaki Endo. CRISPR/Cas9-mediated targeted mutagenesis in grape, PLOS ONE, 2017, Volume 12, Issue 5, DOI: 10.1371/journal.pone.0177966