An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice

Toda, Erika; Koiso, Narumi; Takebayashi, Arika; Ichikawa, Masaki; Kiba, Takatoshi; Osakabe, Keishi; Osakabe, Yuriko; Sakakibara, Hitoshi; Kato, Norio; Okamoto, Takashi

doi:10.1038/s41477-019-0386-z

Cited by 155 publications

(102 citation statements)

References 34 publications

Supporting

Mentioning

101

Contrasting

Order By: Relevance

“…Given that traditional breeding, including chemically and physically induced mutagenesis, and DNA-based genome editing may introduce off-target mutations, editing in a DNA-free manner via preassembled Cas9 protein-guide RNA (gRNA) ribonucleoproteins (RNPs) is an increasingly popular approach due to higher specificity, and low off-target mutations further alleviating public concerns [124][125][126][127] . RNPs have been adopted in the transformation of protoplasts in some horticultural crops, such as lettuce (Lactuca sativa L.) 128 , petunia 129 , apple and grape 130 , and potato 131 .…”

Section: Transgene-free Genome Editingmentioning

confidence: 99%

“…Recently, in planta particle bombardment has been used to deliver CRISPR/Cas9 DNA into shoot apical meristems, resulting in transgene-free homozygous mutated wheat plants 134 . Moreover, recent efforts have been made to deliver RNPs into embryo cells in maize 135 and wheat 116 by particle bombardment and into zygotes by polyethylene glycol-Ca 2+ -mediated transfection in rice 127 .…”

Section: Transgene-free Genome Editingmentioning

confidence: 99%

See 1 more Smart Citation

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Sapkota

Knaap

2020

Hortic Res

View full text Add to dashboard Cite

Directed breeding of horticultural crops is essential for increasing yield, nutritional content, and consumer-valued characteristics such as shape and color of the produce. However, limited genetic diversity restricts the amount of crop improvement that can be achieved through conventional breeding approaches. Natural genetic changes in cisregulatory regions of genes play important roles in shaping phenotypic diversity by altering their expression. Utilization of CRISPR/Cas editing in crop species can accelerate crop improvement through the introduction of genetic variation in a targeted manner. The advent of CRISPR/Cas-mediated cis-regulatory region engineering (cis-engineering) provides a more refined method for modulating gene expression and creating phenotypic diversity to benefit crop improvement. Here, we focus on the current applications of CRISPR/Cas-mediated cis-engineering in horticultural crops. We describe strategies and limitations for its use in crop improvement, including de novo cis-regulatory element (CRE) discovery, precise genome editing, and transgene-free genome editing. In addition, we discuss the challenges and prospects regarding current technologies and achievements. CRISPR/Cas-mediated cis-engineering is a critical tool for generating horticultural crops that are better able to adapt to climate change and providing food for an increasing world population.

show abstract

Section: Transgene-free Genome Editingmentioning

confidence: 99%

Section: Transgene-free Genome Editingmentioning

confidence: 99%

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Sapkota

Knaap

2020

Hortic Res

View full text Add to dashboard Cite

show abstract

“…While classical breeding requires a long time and much labor to produce new traits, the generation of new crop varieties that are otherwise difficult to cross-breed can be achieved using genome editing. Recent studies report transgene free systems as well as novel RNP methods and the optimization of transformation and tissue culture systems [10,11,13,23,24,25,26], which will further increase the advantages of using CRISPR/Cas9 for the production of novel crop varieties in the future.…”

Section: Discussionmentioning

confidence: 99%

“…When transgenes, including genome editing tools, are integrated into mutant genomes, specific regulations, such as the Cartagena Protocol on Biosafety, must be adhered to, both in terms of public acceptance and to facilitate the commercialization of new crop varieties generated by genome editing [9]. Several methods for transgene-free plant genome editing, including RNP (ribonucleoprotein) technology have recently been developed [10,11,12,13]. Null-segregants, i.e., transgene-free mutant plants, can be segregated out from selfing or back-crossed populations.…”

Section: Introductionmentioning

confidence: 99%

Efficient generation of null-segregant parthenocarpic tomato by CRISPR/Cas9 editing

Hara

Ueta

Hashimoto

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background Genome editing technique is a powerful tool in plant genetic engineering to accelerate the rapid breeding of crop plants. Currently, obtaining transgene-free mutant plants is an important issue. transgene-free mutant plants such as null-segregant, can be segregated out from selfing or back-crossed populations. However, such procedures are time consuming, and, therefore, the development of efficient systems to eliminate transgenes in mutant plants is required. Results CRISPR/Cas9 vectors targeting Sl IAA9 were introduced into the tomato cultivars, Ailsa Craig, Moneymaker, Super Roma and Rio Grande, via Agrobacterium transformation. Bi-allelic mutations were detected at ratios of 9–46% in CRISPR/Cas9-transgenic T0 shoots. Sequence analysis revealed that the bi-allelic mutations generated stop codons by frame-shift of SlIAA9 amino acid sequences, indicating that sliaa9 knockout mutants with parthenocarpic phenotypes were generated in the T0 generation with high efficiency in these cultivars. Subsequently, many null-segregant (transgene free) lines, as confirmed by PCR and Southern blot, were isolated rapidly in the T1 generation by self-pollination. Conclusions The method used in this study is effective in achieving rapid isolation of null-segregants with high efficiency, while adding important traits to various cultivars at the same time. Our developed system would further increase the advantages of using CRISPR/Cas9 for the production of novel crop varieties in the future.

show abstract

“…Agrobacterium-mediated delivery is the most popular method of CRISPR/Cas reagent delivery and accounts for more than 80% of the reported editing events, covering almost all model plant species, major field crops, and vegetable and fruit crops . Other delivery methods are suitable for special purposes but are technically complicated, dependent on expensive equipment, or require further development Zhao et al, 2017;Demirer et al, 2019;Kwak et al, 2019;Toda et al, 2019;Zhang et al, 2019). New CRISPR tools have almost always been first reported in rice (Oryza sativa) rather than in other crop plants, primarily because of the availability of high-efficiency Agrobacterium-mediated rice transformation Chen et al, 2019;Hua et al, 2019;Ren et al, 2019;Zhong et al, 2019).…”

mentioning

confidence: 99%

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize

Zhang

et al. 2019

Plant Physiol.

View full text Add to dashboard Cite

ORCID IDs: 0000-0001-6179-5407 (Y.Zhou.); 0000-0001-7827-8211 (Q.-J.C.).The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPRassociated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens. The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

show abstract

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice

Cited by 155 publications

References 34 publications

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Perspectives of CRISPR/Cas-mediated cis-engineering in horticulture: unlocking the neglected potential for crop improvement

Efficient generation of null-segregant parthenocarpic tomato by CRISPR/Cas9 editing

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize

Contact Info

Product

Resources

About