Application of ZFN for Site Directed Mutagenesis of Rice SSIVa Gene

Jung, Yu‐Jin; Nogoy, Franz Marielle; Lee, Sang‐Kyu; Cho, Yong-Gu; Kang, Kwon-Kyoo

doi:10.1007/s12257-017-0420-9

Cited by 52 publications

(23 citation statements)

References 24 publications

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“…ZFN has been reported as efficient for genome editing and can be used as a new rice breeding technology for variety development (Cantos et al, 2014). Recently, to unveil the SSIVa ( Starch Synthase IVa ) gene function, ZFNs targeting the coding region were used to induce DSBs (Jung et al, 2018). Transgenic plants presented premature stop codons and substitution events, leading to inactivation of the SSIVa gene, low starch content and dwarf phenotypes.…”

Section: Targeted Mutationmentioning

confidence: 99%

“…Recently, genome editing technologies have been widely used in various organisms, including plants. The first techniques applied to rice mutagenesis used a non-specific nuclease ( Fok I ) associated to a DNA-specific domain, comprehend the TALEN (Transcription-like effectors nucleases) (Li et al, 2012; Shan et al, 2013) and ZFN (Zinc-finger nucleases) (Cantos et al, 2014; Jung et al, 2018). CRISPR/Cas system has been widely used to induce targeted genomic editing and has been applied in rice since 2013 (Shan et al, 2013; Jiang et al, 2013, Miao et al, 2013; Feng et al, 2013; Shan et al, 2014).…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

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Mutagenesis in Rice: The Basis for Breeding a New Super Plant

et al. 2019

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The high selection pressure applied in rice breeding since its domestication thousands of years ago has caused a narrowing in its genetic variability. Obtaining new rice cultivars therefore becomes a major challenge for breeders and developing strategies to increase the genetic variability has demanded the attention of several research groups. Understanding mutations and their applications have paved the way for advances in the elucidation of a genetic, physiological, and biochemical basis of rice traits. Creating variability through mutations has therefore grown to be among the most important tools to improve rice. The small genome size of rice has enabled a faster release of higher quality sequence drafts as compared to other crops. The move from structural to functional genomics is possible due to an array of mutant databases, highlighting mutagenesis as an important player in this progress. Furthermore, due to the synteny among the Poaceae, other grasses can also benefit from these findings. Successful gene modifications have been obtained by random and targeted mutations. Furthermore, following mutation induction pathways, techniques have been applied to identify mutations and the molecular control of DNA damage repair mechanisms in the rice genome. This review highlights findings in generating rice genome resources showing strategies applied for variability increasing, detection and genetic mechanisms of DNA damage repair.

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Section: Targeted Mutationmentioning

confidence: 99%

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Mutagenesis in Rice: The Basis for Breeding a New Super Plant

et al. 2019

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“…CRISPR/Cas9 is a tremendous technique for precise and targeted editing of the genome of plants and animals. ZFNs (zinc finger nucleases) and TALEN (transcriptional activator-like effector nuclease) genome editing techniques were established before the CRISPR/Cas9, but because of the simplicity and flexibility, rapidness, multiplexing capacity, high efficiency, and mutation frequency, this system gained worldwide popularity and is widely accepted by researchers [ 26 , 27 ]. The CRISPR/Cas9 technique has been successfully applied in Arabidopsis [ 28 , 29 ], rice [ 28 , 30 , 31 , 32 ], maize [ 33 ], wheat [ 31 , 34 ], sorghum [ 28 ], soybean [ 35 , 36 , 37 ], and tomato [ 38 ], and the resulted mutations are transmitted to the next generations according to the classical inheritance principles [ 39 ].…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations

Nawaz

Usman

Zhao

et al. 2020

IJMS

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In rice, semi-dwarfism is among the most required characteristics, as it facilitates better yields and offers lodging resistance. Here, semi-dwarf rice lines lacking any residual transgene-DNA and off-target effects were generated through CRISPR/Cas9-guided mutagenesis of the OsGA20ox2 gene in a high yielding Basmati rice line, and the isobaric tags for relative and absolute quantification (iTRAQ) strategy was utilized to elucidate the proteomic changes in mutants. The results indicated the reduced gibberellins (GA1 and GA4) levels, plant height (28.72%), and flag leaf length, while all the other traits remained unchanged. The OsGA20ox2 expression was highly suppressed, and the mutants exhibited decreased cell length, width, and restored their plant height by exogenous GA3 treatment. Comparative proteomics of the wild-type and homozygous mutant line (GXU43_9) showed an altered level of 588 proteins, 273 upregulated and 315 downregulated, respectively. The identified differentially expressed proteins (DEPs) were mainly enriched in the carbon metabolism and fixation, glycolysis/gluconeogenesis, photosynthesis, and oxidative phosphorylation pathways. The proteins (Q6AWY7, Q6AWY2, Q9FRG8, Q6EPP9, Q6AWX8) associated with growth-regulating factors (GRF2, GRF7, GRF9, GRF10, and GRF11) and GA (Q8RZ73, Q9AS97, Q69VG1, Q8LNJ6, Q0JH50, and Q5MQ85) were downregulated, while the abscisic stress-ripening protein 5 (ASR5) and abscisic acid receptor (PYL5) were upregulated in mutant lines. We integrated CRISPR/Cas9 with proteomic screening as the most reliable strategy for rapid assessment of the CRISPR experiments outcomes.

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“…With the development of some new molecular biology techniques such as CRISPR/Cas9 (clustered regulatory interspersed short palindromic repeat/CRISPR-associated proteins), a lot of achievements have been made in plants and animals (Gaj et al 2013;Gupta et al 2018). CRISPR/Cas9 technology is widely used to study the gene function and regarded as the third-generation genome editing tool established after ZFN (zinc-finger nucleases) and TALEN (transcription activator-like effector nucleases), based on gRNA (guided RNA)-engineered nucleases, which is most applicable due to their simplicity, efficiency, and versatility (Qi et al 2016;Jung et al 2018). In recent years, CRISPR/Cas9 technology has also made remarkable achievements in the study of rice gene function, and has been widely used in human cells, mice, zebrafish, yeast, fruit flies, nematodes, tobacco, and Arabidopsis thaliana (Feng et al 2013;Jiang et al 2013;Ma et al 2015a, b;Bortesi and Fischer 2015).…”

Section: Introductionmentioning

confidence: 99%

Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage

et al. 2019

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Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/ Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T 0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T 1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.

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Application of ZFN for Site Directed Mutagenesis of Rice SSIVa Gene

Cited by 52 publications

References 24 publications

Mutagenesis in Rice: The Basis for Breeding a New Super Plant

Mutagenesis in Rice: The Basis for Breeding a New Super Plant

CRISPR/Cas9 Directed Mutagenesis of OsGA20ox2 in High Yielding Basmati Rice (Oryza sativa L.) Line and Comparative Proteome Profiling of Unveiled Changes Triggered by Mutations

Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage

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