CRISPR/Cas9‐mediated efficient targeted mutagenesis in grape in the first generation

Wang, Xianhang; Tu, Mingxing; Wang, Dejun; Liu, Jianwei; Li, Yajuan; Li, Zhi; Wang, Yuejin

doi:10.1111/pbi.12832

Cited by 281 publications

(180 citation statements)

References 81 publications

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“…The different DNA cleavage efficiency at both target sites is also possibly related it. In grape, variation in mutation frequency among four targets seems to result in different frequency of large deletions between them (Wang et al ., 2017b). Accumulating more data is definitely needed to identify the key factors determining the efficiency of large fragment deletion.…”

Section: Discussionmentioning

confidence: 99%

“…The CRISPR/Cas9 system has been utilized for targeted genomic alternations in many plants, including Arabidopsis thaliana (Lowder et al ., ), Nicotiana benthamiana (Li et al ., ), rice (Shan et al ., ), wheat (Li et al ., ), maize (Feng et al ., ), orange (Jia and Nian, ), apple (Nishitani et al ., ), tomato (Pan et al ., ), grape (Wang et al ., ) and Populus (Fan et al ., ). However, the mutagenesis frequencies of the CRISPR/Cas9 system can vary significantly in plants even within the same species (e.g.…”

Section: Introductionmentioning

confidence: 99%

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Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Wang

et al. 2018

Plant Biotechnology Journal

136

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SummaryKiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Wang

et al. 2018

Plant Biotechnology Journal

136

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“…Interestingly, older leaves possess higher ratio of mutant cells, probably due to the accumuation of DSBs or decreased DSBs repair in older leaves. A very recent study edited the VvWRKY52 transcription factor, which plays a role in biotic stress response (Wang et al ). Proembryonal masses derived from embryo calli were infected with agrobacterium containing the CRISPR/Cas9 construct.…”

Section: Overview Of Fruit Crop Genome Editingmentioning

confidence: 99%

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Zhou¹,

Wang

et al. 2019

JIPB

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Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9‐base‐editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene‐free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer‐friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.

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“…Deletion of the entire PthA4 binding element from both LOB1 alleles resulted in significant citrus canker tolerance [77]. In grapes, CRISPR/Cas9-mediated knockout of WRKY52, encoding a transcription factor related to biotic stress responses, enhanced resistance to Botrytis cinerea [78]. Four gRNAs were designed for different regions in the first exon of WRKY52 to guide the Cas9 nuclease.…”

Section: Genome Editing In Fruit Cropsmentioning

confidence: 99%

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

Corte

Mahmoud

Moraes

et al. 2019

Plants

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Horticultural crops, including fruit, vegetable, and ornamental plants are an important component of the agriculture production systems and play an important role in sustaining human life. With a steady growth in the world's population and the consequent need for more food, sustainable and increased fruit and vegetable crop production is a major challenge to guarantee future food security. Although conventional breeding techniques have significantly contributed to the development of important varieties, new approaches are required to further improve horticultural crop production. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a valuable genome-editing tool able to change DNA sequences at precisely chosen loci. The CRISPR/Cas9 system was developed based on the bacterial adaptive immune system and comprises of an endonuclease guided by one or more single-guide RNAs to generate double-strand breaks. These breaks can then be repaired by the natural cellular repair mechanisms, during which genetic mutations are introduced. In a short time, the CRISPR/Cas9 system has become a popular genome-editing technique, with numerous examples of gene mutation and transcriptional regulation control in both model and crop plants. In this review, various aspects of the CRISPR/Cas9 system are explored, including a general presentation of the function of the CRISPR/Cas9 system in bacteria and its practical application as a biotechnological tool for editing plant genomes, particularly in horticultural crops.

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CRISPR/Cas9‐mediated efficient targeted mutagenesis in grape in the first generation

Cited by 281 publications

References 81 publications

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

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