Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection

Lee, Myoung Hui; Lee, Ji-Young; Choi, Seung Ah; Kim, Ye-Sol; Koo, Ok Jae; Ahn, Woo Seok; Jie, Eun Yee; Kim, Suk Weon

doi:10.1007/s11816-020-00645-2

Cited by 38 publications

(29 citation statements)

References 25 publications

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“…In addition, electroporation was used for Chlamydomonas reinhardtii transformation, and up to 1.1% editing efficiency was achieved ( Baek et al., 2016 ). Recently, electro-transfection has also been demonstrated in cabbage, and editing efficiency as high as 3.4% was obtained ( Lee et al., 2020 ). This method may improve the regeneration ability of protoplasts and increase editing efficiency compared with the PEG method.…”

Section: Rnp Delivery Into Plantsmentioning

confidence: 99%

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CRISPR ribonucleoprotein-mediated genetic engineering in plants

Zhang

Iaffaldano

2021

Plant Communications

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CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement. Commonly used Agrobacterium - or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis. Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site. Genetic engineering using CRISPR ribonucleoproteins (RNPs) has become an attractive approach with many advantages: DNA/transgene-free editing, minimal off-target effects, and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency. Although RNP-mediated genetic engineering has been demonstrated in many plant species, its editing efficiency remains modest, and its application in many species is limited by difficulties in plant regeneration and selection. In this review, we summarize current developments and challenges in RNP-mediated genetic engineering of plants and provide future research directions to broaden the use of this technology.

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Section: Rnp Delivery Into Plantsmentioning

confidence: 99%

“…capitata cv. Dongbok) Phytoene desaturase 1 (PDS1) Cas9 protoplast PEG targeted deep sequencing 1.8% Lee et al. (2020) electro-transfection 0%–3.4% Chinese cabbage ( Brassica rapa subsp.…”

Section: Rnp Applications In Plant Genome Editingmentioning

confidence: 99%

CRISPR ribonucleoprotein-mediated genetic engineering in plants

Zhang

Iaffaldano

2021

Plant Communications

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“…Woo et al (2015) succeeded in delivering CRISPR-Cas9 RNPs into lettuce protoplasts by PEG-mediated transfection and, subsequently, regenerated plants with intended mutations from the protoplasts (Woo et al, 2015). Recently, it has been suggested that electroporation is more efficient in RNP delivery to protoplasts than PEG-mediated transfection in cabbage based on the results of phytoene desaturase1 (PDS1) sgRNA delivery, which may be due to lower chemical toxicity (Lee et al, 2020). It has also been found that genome editing of upstream open reading frame (uORF) enabled the modulation of translation of mRNA.…”

Section: Leafy Vegetablesmentioning

confidence: 99%

“…alboglabra), and cabbage (Brassica oleracea var. capitata; Ma et al, 2019a,b;Lee et al, 2020), and PDS mutants were detected by their albino phenotype. CRISPR-Cas9 was also used to induce mutations in the carotenoid isomerase gene (BoaCRTISO) of Chinese kale (Sun et al, 2020 (Deng et al, 2011;Jeong et al, 2019).…”

Section: Other Leafy Vegetablesmentioning

confidence: 99%

Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review

Kim

Kang²,

Yang

et al. 2021

Front. Plant Sci.

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The emergence of genome-editing technology has allowed manipulation of DNA sequences in genomes to precisely remove or replace specific sequences in organisms resulting in targeted mutations. In plants, genome editing is an attractive method to alter gene functions to generate improved crop varieties. Genome editing is thought to be simple to use and has a lower risk of off-target effects compared to classical mutation breeding. Furthermore, genome-editing technology tools can also be applied directly to crops that contain complex genomes and/or are not easily bred using traditional methods. Currently, highly versatile genome-editing tools for precise and predictable editing of almost any locus in the plant genome make it possible to extend the range of application, including functional genomics research and molecular crop breeding. Vegetables are essential nutrient sources for humans and provide vitamins, minerals, and fiber to diets, thereby contributing to human health. In this review, we provide an overview of the brief history of genome-editing technologies and the components of genome-editing tool boxes, and illustrate basic modes of operation in representative systems. We describe the current and potential practical application of genome editing for the development of improved nutritious vegetables and present several case studies demonstrating the potential of the technology. Finally, we highlight future directions and challenges in applying genome-editing systems to vegetable crops for research and product development.

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“…RNPs enable immediate transient gene editing with reduced off-target effects (Kim et al , 2014; Yip, 2020). Cas9-sgRNA RNPs have been shown to efficiently edit the genomes of human and animal cells (Cho et al , 2013; Kim et al , 2014; Chaverra-Rodriguez et al , 2018; Chen et al , 2019), plant cells (Park et al , 2019; Lee et al , 2020) and various fungi (Foster et al , 2018; Zou et al , 2020). In A. pullulans , CRISPR mediated genome editing was previously performed using plasmids (Zhang et al , 2019) but RNPS have not yet been used.…”

Section: Introductionmentioning

confidence: 99%

Fast and efficient CRISPR-mediated genome editing in Aureobasidium pullulans using Cas9 ribonucleoproteins

Kreuter

Stark

Mach

et al. 2021

Preprint

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Aureobasidium pullulans is a ubiquitous, polyextremotolerant, yeast-like ascomycete used for the industrial production of pullulan and other products and as biocontrol agent in the agriculture. Its application potential and its wide-spread occurrence make A. pullulans an interesting study object. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on A. pullulans. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs). We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting ura3 (encoding for orotidine-5'-phosphate decarboxylase), praics (encoding for phosphoribosyl aminoimidazole-succinocarboxamide synthase) and asl (encoding for arginine succinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the ura3 deficiency. Further, we show that the usage of RNPs can boost the homologous recombination rate up to nearly 100%, even when using only 20bp long homologous flanks. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in A. pullulans.

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Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection

Cited by 38 publications

References 25 publications

CRISPR ribonucleoprotein-mediated genetic engineering in plants

CRISPR ribonucleoprotein-mediated genetic engineering in plants

Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review

Fast and efficient CRISPR-mediated genome editing in Aureobasidium pullulans using Cas9 ribonucleoproteins

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