Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins

Fan, Yueting; Xin, Shichao; Dai, Xuemei; Yang, Xianfeng; Hong, Huang; Yue, Hua

doi:10.1016/j.indcrop.2020.112146

Cited by 60 publications

(23 citation statements)

References 34 publications

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“…Genome editing with RNPs has been successfully applied in several species using protoplasts [39,[67][68][69] and immature embryos [70,71]. In our study we have successfully edited P. radiata somatic embryogenic cells and produced plantlets using RNPs.…”

Section: Discussionmentioning

confidence: 94%

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

Poovaiah

Phillips

Geddes

et al. 2020

Preprint

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BackgroundTo meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies.ResultsWe evaluated U6 snRNA promoters from three different species that were able to drive expression of guide RNA (gRNA) for CRISPR/Cas9 genome editing in P. radiata. Using a single-copy cell wall gene GUX1 as a target, we have demonstrated genome editing using CRISPR/Cas9 in somatic embryogenic tissue and plantlets derived from the edited tissue. We generated biallelic INDELs with an efficiency of 15% using a single gRNA. Twelve percent of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We also assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33%, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic.Conclusion We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using DNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

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Section: Discussionmentioning

confidence: 94%

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

Poovaiah

Phillips

Geddes

et al. 2020

Preprint

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“…Although a large number of genomic data and transcriptome data of rubber tree can be obtained [9][10][11], but few functional genes have been proved in rubber tree [12][13][14][15]. Functional gene analysis is still a major challenge lacking e cient and reliable genetic transformation system [15,16]. In this study, we examined Agrobacterium-mediated TRV-based VIGS in rubber tree.…”

Section: Discussionmentioning

confidence: 99%

“…To date, rubber biosynthesis pathway has been well characterized [8], the whole-genome data and transcriptome database became available in rubber tree [9][10][11], but only a few NR biosynthesis-related genes functions are evaluated by transgenic rubber tree due to low genetic transformation e ciency [12][13][14][15]. The development of an e cient and reliable rubber tree transformation system is still challenging [15,16].…”

Section: Introductionmentioning

confidence: 99%

Tobacco rattle virus -induced gene silencing in Hevea brasiliensis

Wang

Zhu

et al. 2020

Preprint

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BackgroundSince it is very difficult to obtain gene knockouts in rubber tree (Hevea Brasiliensis) due to low genetic transformation efficiency. Virus-induced gene silencing (VIGS) is a powerful gene silencing tool that has been intensively applied in plant. Up to now, the application of VIGS in rubber tree has not yet been reported.ResultsHevea brasiliensis phytoene desaturase (HbPDS) was identified in H. brasiliensis genome. The prediction of small interfering RNAs (siRNAs) from HbPDS and the silencing gene fragment (SGF) were predicted and a length of 409 bp SGF was chosen to be tested. We show that the tobacco rattle virus (TRV) -VIGS is able to induce effective HbPDS silencing in rubber tree. The TRV-VIGS system has the potential for functional gene studies in rubber tree.ConclusionsThis is the first time to report VIGS in rubber tree. The present TRV-VIGS method could be further applied to produce gene silenced rubber tree plants, to advance functional gene of rubber tree. The applied TRV-VIGS method will achieve deeper underground into the natural rubber biosynthesis and regulation in this important rubber-producing plant.

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“…Biomacromolecules can be delivered into the plant protoplast in the presence of PEG. A large number of works have been performed to optimize PEG-mediated RNP delivery into various plant and fungi protoplast cells 85 , 132 - 141 . In allusion to the plant species whose protoplasts are difficult to be isolated and cultured, researchers bypassed the problem by direct delivery of RNP into plant zygotes which were produced by in vitro fertilization of isolated gametes 142 .…”

Section: Physical Approaches For Rnp Deliverymentioning

confidence: 99%

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

Song¹,

Shen²,

Li³

et al. 2021

Theranostics

274

184

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CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.

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Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins

Cited by 60 publications

References 34 publications

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

Tobacco rattle virus -induced gene silencing in Hevea brasiliensis

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

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Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins

Cited by 60 publications

References 34 publications

Genome Editing With CRISPR/Cas9&nbsp;in Pinus Radiata (D. Don)

Genome Editing With CRISPR/Cas9&nbsp;in Pinus Radiata (D. Don)

Tobacco rattle virus -induced gene silencing in Hevea brasiliensis

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

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Product

Resources

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Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)