Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

Yue, Jin-Jun; Yuan, Jiangang; Wu, Fan; Yuan, Yasheng; Cheng, Qiao-Wei; Hsu, Chen-Tran; Lin, Chun-Shin

doi:10.3389/fgeed.2021.717017

Cited by 52 publications

(34 citation statements)

References 86 publications

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“…Plant “protoplasts” (whose name originates from the ancient Greek prōtóplastos, meaning “first-formed”) are whole plant cells without cell walls, and have been widely used in plant science research and crop breeding. Protoplast transformation/transfection involves the direct delivery of plasmid DNA to individual protoplasts using polyethylene glycol (PEG), electroporation or other methods [ 155 ]. Protoplast transformation has several benefits, including: delivery of multiple plasmids with high levels of cotransformation, no binary vector required, and providing the ability to check CRISPR construct efficiency in a short time.…”

Section: Enabling Technologiesmentioning

confidence: 99%

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Thomson

Biswas

Tsakirpaloglou

et al. 2022

IJMS

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Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.

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Section: Enabling Technologiesmentioning

confidence: 99%

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Thomson

Biswas

Tsakirpaloglou

et al. 2022

IJMS

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show abstract

“…Another topic commonly associated with genome editing and CRISPR tools is using protoplasts (plant cells without cell walls) as a rapid validation system. It provides a platform to test the mutagenesis efficiency of RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins before the full-scale transformation in the host plant [131]. The popularity of this approach is reflected in the network map in which the keyword "protoplast" is present near "site-directed mutagenesis", "crispr/cas", and "transgene" nodes.…”

Section: Vosviewer Bibliometric Analysismentioning

confidence: 99%

Genome Editing for Sustainable Crop Improvement and Mitigation of Biotic and Abiotic Stresses

Hamdan¹,

Karlson²,

Teoh³

et al. 2022

Preprint

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Climate change poses a serious threat to global agricultural activity and food production. To address this issue, plant genome editing technologies have been developed to provide an alternative solution for crop improvement. Unlike conventional breeding techniques (e.g., selective breeding and mutation breeding), modern genome editing tools offer more targeted and specific alterations of the plant genome to produce crops with desired traits, such as higher yield and/or stronger resilience to the changing environment. In this review, we discuss the current development and future applications of genome editing technologies in mitigating the impacts of biotic and abiotic stresses on agriculture. We focus specifically on the CRISPR/Cas system, which has been the center of attention in the last few years as a revolutionary genome-editing tool in various species. We also conducted a bibliographic analysis on CRISPR-related papers published from 2012 to 2021 (10 years) to identify trends and possible gaps in the CRISPR/Cas-related plant research. In addition, this review article outlines the current shortcomings and challenges of employing genome editing technologies in agriculture with notes on future prospective. We believe combining conventional and more innovative technologies in agriculture would be the key to optimizing crop improvement beyond the limitations of traditional agricultural practices.

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“…Surprisingly, Andersson et al (2018) reported that a large proportion of the regenerated plants contained inserts at the target site, containing either random fragments of potato chromosomal DNA or originating from the DNA template used to synthesize the gRNA. Despite the success of PEG-mediated delivery of RNPs in certain transformation-recalcitrant species, few plant species have been satisfactorily regenerated from protoplasts ( Yue et al, 2021 ). Moreover, genome instability caused by protoplast regeneration is not infrequent ( Fossi et al, 2019 ).…”

Section: Ribonucleoproteinsmentioning

confidence: 99%

“…Moreover, genome instability caused by protoplast regeneration is not infrequent ( Fossi et al, 2019 ). Due to a lack of well-established and species-specific protoplast isolation and regeneration techniques, especially for monocotyledonous plants, the adoption of PEG-mediated RNP genome editing has been limited thus far ( Yue et al, 2021 ). Other useful strategies for the delivery of genes or proteins to mammalian cells, such as electroporation and lipofection, have also been tested in plants.…”

Section: Ribonucleoproteinsmentioning

confidence: 99%

Non-GM Genome Editing Approaches in Crops

2021

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CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

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Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

Cited by 52 publications

References 86 publications

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Genome Editing for Sustainable Crop Improvement and Mitigation of Biotic and Abiotic Stresses

Non-GM Genome Editing Approaches in Crops

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