CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)

Wan, Dongyan; Guo, Yifeng; Cheng, Yuan; Yang, Huanming; Xiao, Shaobo; Wang, Yuejin; Wen, Ying-Qiang

doi:10.1038/s41438-020-0339-8

Cited by 138 publications

(71 citation statements)

References 51 publications

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“…‘Seishi’ × M. pumila ) 23 – 25 , orange ( Citrus sinensis (L.) Osbeck) 26 , 27 , kiwifruit ( Actinidia deliciosa ) 28 , 29 , and grapevine ( Vitis vinifera ) 12 , 30 – 33 . However, the extent of off-target mutations in CRISPR/Cas9-edited fruit trees is still not completely examined and has mainly been investigated using target sequencing 12 , 31 , 32 .…”

Section: Introductionmentioning

confidence: 99%

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

Wang

et al. 2021

Hortic Res

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The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

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Section: Introductionmentioning

confidence: 99%

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

Wang

et al. 2021

Hortic Res

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“…In the past eight years, there have been many reports of creating genome-edited plants with resistance to viral, bacterial, and fungal diseases in different plant species using CRISPR/Cas technology ( Zhang et al, 2021 ). These include CRISPR/Cas9 knock-outs of the following genes: mlo for plant resistance to powdery mildew in wheat ( Wang et al, 2014 ), tomato ( Nekrasov et al, 2017 ; Martínez et al, 2020 ) and grapevine ( Wan et al, 2020 ), pmr4 for plant resistance to powdery mildew in tomato (Santillán Martínez et al, 2020), 14-3-3 gene for resistance to Verticillium dahlia in cotton ( Zhang ZN et al, 2018 ), crt1a for resistance to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape ( Pröbsting et al, 2020 ), OsERF922 for resistance to Magnaporthe oryzae in rice ( Wang et al, 2016 ), Clpsk1 for resistance to Fusarium oxysporum f. sp. niveum in watermelon ( Zhang et al, 2020 ), eif4e for resistance to Cucumber vein yellowing virus , Zucchini yellow mosaic virus , and Papaya ring spot mosaic virus‐W in cucumber ( Chandrasekaran et al, 2016 ), CsWRKY22 for resistance to Xanthomonas citri subsp.…”

Section: Crispr/cas Is a Robust And Powerful Tool For Precision Brementioning

confidence: 99%

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Brant

Budak

et al. 2021

J. Zhejiang Univ. Sci. B

110

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Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

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“…Recently, a genome-wide survey of suitable sites for CRISPR/Cas9 genome editing has been conducted in grapevine ( Wang et al, 2016 ) and successful attempts to actually generate genome-edited grapevine have been reported ( Ren et al, 2016 ; Wang et al, 2016 ). Although the latter were just merely proof of concept attempts, Wan et al (2020) reported this technology to generate grapevine plants with enhanced powdery mildew resistance through Mlo gene edition. The authors reported a 38.5% successful gene edition rate, a value lower to those previously reported in rice (84.3% on average) but comparable to those obtained in Arabidopsis (35.6% on average) ( Ma et al, 2015 ).…”

Section: Controlling the Genome And Its Expressionmentioning

confidence: 99%

Molecular Tools for Adapting Viticulture to Climate Change

2021

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Adaptation of viticulture to climate change includes exploration of new geographical areas, new training systems, new management practices, or new varieties, both for rootstocks and scions. Molecular tools can be defined as molecular approaches used to study DNAs, RNAs, and proteins in all living organisms. We present here the current knowledge about molecular tools and their potential usefulness in three aspects of grapevine adaptation to the ongoing climate change. (i) Molecular tools for understanding grapevine response to environmental stresses. A fine description of the regulation of gene expression is a powerful tool to understand the physiological mechanisms set up by the grapevine to respond to abiotic stress such as high temperatures or drought. The current knowledge on gene expression is continuously evolving with increasing evidence of the role of alternative splicing, small RNAs, long non-coding RNAs, DNA methylation, or chromatin activity. (ii) Genetics and genomics of grapevine stress tolerance. The description of the grapevine genome is more and more precise. The genetic variations among genotypes are now revealed with new technologies with the sequencing of very long DNA molecules. High throughput technologies for DNA sequencing also allow now the genetic characterization at the same time of hundreds of genotypes for thousands of points in the genome, which provides unprecedented datasets for genotype-phenotype associations studies. We review the current knowledge on the genetic determinism of traits for the adaptation to climate change. We focus on quantitative trait loci and molecular markers available for developmental stages, tolerance to water stress/water use efficiency, sugar content, acidity, and secondary metabolism of the berries. (iii) Controlling the genome and its expression to allow breeding of better-adapted genotypes. High-density DNA genotyping can be used to select genotypes with specific interesting alleles but genomic selection is also a powerful method able to take into account the genetic information along the whole genome to predict a phenotype. Modern technologies are also able to generate mutations that are possibly interesting for generating new phenotypes but the most promising one is the direct editing of the genome at a precise location.

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CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)

Cited by 138 publications

References 51 publications

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Molecular Tools for Adapting Viticulture to Climate Change

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