Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene

Gonzalez, Matías Nicolás; Massa, G; Andersson, Mariette; Oneto, Cecilia Andrea Décima; Turesson, Helle; Storani, Leonardo; Olsson, Niklas; Fält, Ann-Sofie; Hofvander, Per; Feingold, Sérgio Enrique

doi:10.1007/s11240-020-02008-9

Cited by 30 publications

(24 citation statements)

References 56 publications

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“…The CRISPR/Cas9 system has been successfully applied in various plant species for site-specific base modifications through various genetic transformation methods with varying editing efficiency. It has been observed that the editing efficiency of 9.6%, 18.4%, and 31.9% can be obtained in the edited lines depending on the Agrobacterium -mediated transformation, ribonucleoprotein complexes, and transient expression in protoplasts respectively [ 21 ]. Another advancement of the CRISPR/Cas9 genome editing, the Transiently Expressed CRISPR/Cas DNA (TECCDNA) is a simple and efficient genome-editing approach in which mutant plants are regenerated after transient expression of CRISPR/Cas9 DNA [ 22 ].…”

Section: Genome Editing Methodsmentioning

confidence: 99%

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Kaur

Manchanda

Kalia

et al. 2021

IJMS

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Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

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Section: Genome Editing Methodsmentioning

confidence: 99%

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Kaur

Manchanda

Kalia

et al. 2021

IJMS

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“…Since this first report, RNPs have been successfully used to mediate potato genome editing using the protoplasts transfection and regeneration system, with editing efficiencies ranging from 27 to 68% ( González et al, 2020 ) and 52 to 72% ( Zhao et al, 2021 ), varying with the targeted genes and the design of RNP components. Protoplasts provide a suitable platform for genome-editing reagents delivery and may in some cases represent even a more efficient strategy when compared to Agrobacterium -mediated transformation ( González et al, 2021 ). Even though it represents a promising strategy to mediate transgene-free genome editing in potato, the use of RNP in protoplasts present some important aspects to consider.…”

Section: Genome Editing In Potatomentioning

confidence: 99%

“…Even though it represents a promising strategy to mediate transgene-free genome editing in potato, the use of RNP in protoplasts present some important aspects to consider. For instance, DNA traces remaining in the assembled RNPs may led to unintended foreign DNA insertions in the regenerated plants, with different frequencies ( Andersson et al, 2018 ; González et al, 2021 ; Zhao et al, 2021 ). A possible origin of such traces is the DNA molecules employed for the in vitro transcription of sgRNAs, before RNP assembly.…”

Section: Genome Editing In Potatomentioning

confidence: 99%

State of the Art of Genetic Engineering in Potato: From the First Report to Its Future Potential

et al. 2022

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Potato (Solanum tuberosum L.) is a crop of world importance that produces tubers of high nutritional quality. It is considered one of the promising crops to overcome the challenges of poverty and hunger worldwide. However, it is exposed to different biotic and abiotic stresses that can cause significant losses in production. Thus, potato is a candidate of special relevance for improvements through conventional breeding and biotechnology. Since conventional breeding is time-consuming and challenging, genetic engineering provides the opportunity to introduce/switch-off genes of interest without altering the allelic combination that characterize successful commercial cultivars or to induce targeted sequence modifications by New Breeding Techniques. There is a variety of methods for potato improvement via genetic transformation. Most of them incorporate genes of interest into the nuclear genome; nevertheless, the development of plastid transformation protocols broadened the available approaches for potato breeding. Although all methods have their advantages and disadvantages, Agrobacterium-mediated transformation is the most used approach. Alternative methods such as particle bombardment, protoplast transfection with polyethylene glycol and microinjection are also effective. Independently of the DNA delivery approach, critical steps for a successful transformation are a rapid and efficient regeneration protocol and a selection system. Several critical factors affect the transformation efficiency: vector type, insert size, Agrobacterium strain, explant type, composition of the subculture media, selective agent, among others. Moreover, transient or stable transformation, constitutive or inducible promoters, antibiotic/herbicide resistance or marker-free strategies can be considered. Although great efforts have been made to optimize all the parameters, potato transformation protocols are highly genotype-dependent. Genome editing technologies provide promising tools in genetic engineering allowing precise modification of targeted sequences. Interestingly, transient expression of genome editing components in potato protoplasts was reported to generate edited plants without the integration of any foreign DNA, which is a valuable aspect from both a scientific and a regulatory perspective. In this review, current challenges and opportunities concerning potato genetic engineering strategies developed to date are discussed. We describe their critical parameters and constrains, and the potential application of the available tools for functional analyses or biotechnological purposes. Public concerns and safety issues are also addressed.

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“…After delivering the complex into the cell nucleus, RNP is rapidly degraded, thus avoiding potential off-target effects ( Kim J.-S. et al, 2015 , 2017 ; Subburaj et al, 2016 ). Moreover, for cellular toxicity associated with long-term expression of Cas and/or integration of exogenous DNA, the RNP complex approach may represent a good choice due to the transient and stable transfection in the plant cell ( González et al, 2021 ). On the other hand, as this strategy does not use selection marker genes, the screening of edited plants with desirable phenotypes may become more laborious and costly.…”

Section: Genome Editing Technology Focuses On Crispr/cas Technologymentioning

confidence: 99%

CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture

Távora

Diniz

Rêgo-Machado

et al. 2022

Front. Bioeng. Biotechnol.

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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe.

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Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene

Cited by 30 publications

References 56 publications

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

State of the Art of Genetic Engineering in Potato: From the First Report to Its Future Potential

CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture

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