Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene

Nakayasu, Masaru; Akiyama, Ryota; Lee, Hyoung Jae; Osakabe, Keishi; Osakabe, Yuriko; Watanabe, Bunta; Sugimoto, Yukihiro; Umemoto, Naoyuki; Saito, Kazuki; Muranaka, Toshiya; Mizutani, Masaharu

doi:10.1016/j.plaphy.2018.04.026

Cited by 167 publications

(71 citation statements)

References 35 publications

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“…In some plants, wild‐type sequence was detectable while in others transformed with the same construct all targets were mutated. This kind of variability has been observed previously for Cas9‐mediated mutagenesis of potatoes (Andersson et al ., ; Kusano et al ., ; Nakayasu et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Tuncel

Corbin

Ahn‐Jarvis

et al. 2019

Plant Biotechnology Journal

117

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Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

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Section: Discussionmentioning

confidence: 97%

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Tuncel

Corbin

Ahn‐Jarvis

et al. 2019

Plant Biotechnology Journal

117

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“…To achieve this goal, TALEN and CRISPR/Cas9 were used to knockout the SSR2 gene encoding for sterol side chain reductase 2 and the St16DOX gene encoding for the steroid 16α-hydroxylase in the SGA biosynthetic pathway. This method prevented SGA accumulation in potato tuber and hairy roots, respectively (Nakayasu et al, 2018;Yasumoto et al, 2019). Meanwhile, high amylopectin (amylosefree) starch has been an important common trait in staple crops due its commercial value in the food and manufacturing paper industries.…”

Section: Staple Cropsmentioning

confidence: 99%

Improving Nutritional and Functional Quality by Genome Editing of Crops: Status and Perspectives

2020

Front. Plant Sci.

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Genome-editing tools including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR) system have been applied to improve the quality of staple, oilseed, and horticultural crops with great accuracy and efficiency compared to conventional breeding. In particular, the CRISPR method has proven to be a feasible, cost-effective and versatile tool allowing precise and efficient editing of plant genomes in recent years, showing great potential in crop improvement. Until now, various genome-edited crops with enhanced commercial value have been developed by not only global companies but also small laboratories in universities, suggesting low entry barriers with respect to manpower and capital. In this study, we review the current applications of genome editing technologies to improve the nutritional and functional quality and preferred traits of various crops. Combining this rapidly advancing genome-editing technology and conventional breeding will greatly extend the potential of genome-edited crops and their commercialization.

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“…The knockout tomato hairy roots for each of SlS5αR1 and SlS5αR2 were generated by targeted genome editing with the CRISPR/Cas9 system. We used the CRISPR/Cas9 binary vector pMgP237-2A-GFP to express multiplex gRNAs (Hashimoto et al 2018;Nakayasu et al 2018). To design a gRNA target with low off-target effect in SlS5αR1 and SlS5αR2, we conducted in silico analyses using the Web tool Design sgRNAs for CRISPRko (https://portals.broadinstitute.…”

Section: Construction Of Crispr/cas9 Vectorsmentioning

confidence: 99%

“…To verify the contribution of SlS5αR1 and SlS5αR2 in α-tomatine biosynthesis in vivo, SlS5αR1 and SlS5αR2 were each independently disrupted using CRISPR/Cas9-mediated genome editing in tomato hairy roots. In this study, we used a CRISPR/Cas9 binary vector designated as pMgP237-2A-GFP that permits expression of multiplex gRNAs by a pre-tRNA processing mechanism (Hashimoto et al 2018;Nakayasu et al 2018). To design the gRNAs specific to SlS5αR1 and to SlS5αR2, we conducted in silico analysis using Design sgRNAs for CRISPRko and Cas-OT software (Xiao et al 2014).…”

Section: Crispr/cas9-mediated Genome Editing Of Sls5αr1 or Sls5αr2 Inmentioning

confidence: 99%

Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

Akiyama

Lee

Nakayasu

et al. 2019

Plant Biotechnology

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α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3βHSD is involved in the three reactions except for C5α reduction, and in the present study, we aimed to elucidate the gene responsible for the C5α reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5αR1 and SlS5αR2, which show high homology with DET2, a brassinosteroid 5α reductase of Arabidopsis thaliana. The expression pattern of SlS5αR2 is similar to those of SGA biosynthetic genes, while SlS5αR1 is ubiquitously expressed, suggesting the involvement of SlS5αR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5αR1 and SlS5αR2 catalyze the reduction of tomatid-4-en-3-one at C5α to yield tomatid-3-one. Then, SlS5αR1-or SlS5αR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5αR2-knockout hairy roots, the α-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of α-tomatine was observed in the SlS5αR1-knockout hairy root. These results indicate that SlS5αR2 is responsible for the C5α reduction in α-tomatine biosynthesis and that SlS5αR1 does not significantly contribute to α-tomatine biosynthesis.

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Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene

Cited by 167 publications

References 35 publications

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Improving Nutritional and Functional Quality by Genome Editing of Crops: Status and Perspectives

Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

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