Efficient Genome Editing in Apple Using a CRISPR/Cas9 system

Nishitani, Chikako; Hirai, Narumi; Komori, Sadao; Wada, Masato; Okada, Kazuma; Osakabe, Keishi; Yamamoto, Takatsugu; Osakabe, Yuriko

doi:10.1038/srep31481

Cited by 278 publications

(182 citation statements)

References 29 publications

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“…CRISPR/Cas9 has been successfully employed in perennial species including apple (Nishitani et al, 2016) and sweet orange ( Citrus sinensis ) (Jia and Wang, 2014). Clearly, incorporation of desirable traits from wild relatives into perennial crops is not limited to MAS, but can also be achieved through GM.…”

Section: Genomic Resources and Limitations: Mapping And Breedingmentioning

confidence: 99%

Exploiting Wild Relatives for Genomics-assisted Breeding of Perennial Crops

2017

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Perennial crops are vital contributors to global food production and nutrition. However, the breeding of new perennial crops is an expensive and time-consuming process due to the large size and lengthy juvenile phase of many species. Genomics provides a valuable tool for improving the efficiency of breeding by allowing progeny possessing a trait of interest to be selected at the seed or seedling stage through marker-assisted selection (MAS). The benefits of MAS to a breeder are greatest when the targeted species takes a long time to reach maturity and is expensive to grow and maintain. Thus, MAS holds particular promise in perennials since they are often costly and time-consuming to grow to maturity and evaluate. Well-characterized germplasm that breeders can tap into for improving perennials is often limited in genetic diversity. Wild relatives are a largely untapped source of desirable traits including disease resistance, fruit quality, and rootstock characteristics. This review focuses on the use of genomics-assisted breeding in perennials, especially as it relates to the introgression of useful traits from wild relatives. The identification of genetic markers predictive of beneficial phenotypes derived from wild relatives is hampered by genomic tools designed for domesticated species that are often ill-suited for use in wild relatives. There is therefore an urgent need for better genomic resources from wild relatives. A further barrier to exploiting wild diversity through genomics is the phenotyping bottleneck: well-powered genetic mapping requires accurate and cost-effective characterization of large collections of diverse wild germplasm. While genomics will always be used in combination with traditional breeding methods, it is a powerful tool for accelerating the speed and reducing the costs of breeding while harvesting the potential of wild relatives for improving perennial crops.

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Section: Genomic Resources and Limitations: Mapping And Breedingmentioning

confidence: 99%

Exploiting Wild Relatives for Genomics-assisted Breeding of Perennial Crops

2017

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“…CRISPR/Cas9 editing tools are efficiently exploited for gene mutation, repression, activation and epigenome editing in several model and crop plants, such as Arabidopsis, tobacco, rice, sorghum, maize, wheat, poplar, tomato, soya bean, petunia, citrus and recently grape and apple (Nishitani et al, 2016; Ren et al, 2016; Song et al, 2016). Meanwhile CRISPR/Cas9 RNPs DNA-free genome editing tools are successfully demonstrated in Arabidopsis , tobacco, lettuce, rice, petunia, and recently in wheat (Woo et al, 2015; Subburaj et al, 2016; Zhang et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

et al. 2016

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The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

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“…There have been limited reports of successful CRISPR/Cas9 genome editing in Rosaceae plants. In apple, CRISPR/Cas9 genome editing was reported to occur in apple protoplasts (Malnoy et al, 2016) and in callus-generated shoots (Nishitani et al, 2016). However, there has been no report of successful inheritance of CRISPR-induced mutations as most of the mutations were generated in somatic cells that could not yield progeny.…”

Section: Introductionmentioning

confidence: 99%

Efficient genome editing of wild strawberry genes, vector development and validation

Zhou

Wang

Liu

2018

Plant Biotechnology Journal

104

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SummaryThe clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR–Cas9 to horticultural plants, especially with regard to germ‐line transmission of targeted mutations. Here, we report high‐efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6‐26 and the wild strawberry U6 promoter FveU6‐2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high‐efficiency genome editing in strawberry. To test germ‐line transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harbouring arf8 homozygous knockout mutations grew considerably faster than wild‐type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance.

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Efficient Genome Editing in Apple Using a CRISPR/Cas9 system

Cited by 278 publications

References 29 publications

Exploiting Wild Relatives for Genomics-assisted Breeding of Perennial Crops

Exploiting Wild Relatives for Genomics-assisted Breeding of Perennial Crops

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

Efficient genome editing of wild strawberry genes, vector development and validation

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