DNA-Free Genome Editing via Ribonucleoprotein (RNP) Delivery of CRISPR/Cas in Lettuce

Choi, Sunmee; Park, Slki; Yoon, Ji-Young; Park, Aiden Y; Choe, Senyon

doi:10.1007/978-1-4939-8991-1_25

Cited by 37 publications

(20 citation statements)

References 10 publications

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“…Through the application of these delivery systems, the DNA-free editing approach has been successfully applied to a number of species. A review by Metje-Sprink et al [ 164 ] reports that researchers have achieved transgene-free editing in N. benthaminiana [ 162 ], Solanum tuberosum [ 165 ], T. aestivum , and Z. mays [ 166 , 167 ], Brassicaceae [ 168 ], O. sativa [ 169 ], Musa acuminata [ 170 ], Lactuca sativa [ 171 ], and Piper nigrum [ 172 ]. However, this method has some drawbacks.…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security

Fiaz

Ahmar

Saeed

et al. 2021

IJMS

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A world with zero hunger is possible only through a sustainable increase in food production and distribution and the elimination of poverty. Scientific, logistical, and humanitarian approaches must be employed simultaneously to ensure food security, starting with farmers and breeders and extending to policy makers and governments. The current agricultural production system is facing the challenge of sustainably increasing grain quality and yield and enhancing resistance to biotic and abiotic stress under the intensifying pressure of climate change. Under present circumstances, conventional breeding techniques are not sufficient. Innovation in plant breeding is critical in managing agricultural challenges and achieving sustainable crop production. Novel plant breeding techniques, involving a series of developments from genome editing techniques to speed breeding and the integration of omics technology, offer relevant, versatile, cost-effective, and less time-consuming ways of achieving precision in plant breeding. Opportunities to edit agriculturally significant genes now exist as a result of new genome editing techniques. These range from random (physical and chemical mutagens) to non-random meganucleases (MegaN), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein system 9 (CRISPR/Cas9), the CRISPR system from Prevotella and Francisella1 (Cpf1), base editing (BE), and prime editing (PE). Genome editing techniques that promote crop improvement through hybrid seed production, induced apomixis, and resistance to biotic and abiotic stress are prioritized when selecting for genetic gain in a restricted timeframe. The novel CRISPR-associated protein system 9 variants, namely BE and PE, can generate transgene-free plants with more frequency and are therefore being used for knocking out of genes of interest. We provide a comprehensive review of the evolution of genome editing technologies, especially the application of the third-generation genome editing technologies to achieve various plant breeding objectives within the regulatory regimes adopted by various countries. Future development and the optimization of forward and reverse genetics to achieve food security are evaluated.

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Section: Challenges and Future Perspectivesmentioning

confidence: 99%

Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security

Fiaz

Ahmar

Saeed

et al. 2021

IJMS

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“…At present, manifold systems have been conceived and tested to isolate transgene-free CRISPRed plants. Despite the delivery of ribonucleoprotein (RNP) complexes via protoplasts transfection produced non-transgenic mutants in Arabidopsis thaliana , tobacco and rice 17 , lettuce 18 and potato 19 , these approaches are limited only to some species due to bottlenecks in the regeneration process for many other crops 20 . Moreover, regeneration of a plant from protoplasts may be risky due to substantial genome instability of in-vitro cultured protoplasts.…”

Section: Discussionmentioning

confidence: 99%

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Costa

Piazza

Pompili

et al. 2020

Sci Rep

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Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

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“…The recently used RNP, a technique for achieving GE plants, includes apple [ 103 ], banana [ 142 ], brassica [ 186 ], capsicum [ 190 ], rice [ 191 ], potato [ 188 ], and lettuce [ 192 ]. In RNP-mediated delivery, Cas9 swiftly becomes degraded after cleaving the target site, thus reducing the off-targets vulnerability and GMO/ethical concerns.…”

Section: Genome Editing In Precision Breedingmentioning

confidence: 99%

Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience

Sattar

Iqbal

Al-Khayri

et al. 2021

Plants

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Fruit trees provide essential nutrients to humans by contributing to major agricultural outputs and economic growth globally. However, major constraints to sustainable agricultural productivity are the uncontrolled proliferation of the population, and biotic and abiotic stresses. Tree mutation breeding has been substantially improved using different physical and chemical mutagens. Nonetheless, tree plant breeding has certain crucial bottlenecks including a long life cycle, ploidy level, occurrence of sequence polymorphisms, nature of parthenocarpic fruit development and linkage. Genetic engineering of trees has focused on boosting quality traits such as productivity, wood quality, and resistance to biotic and abiotic stresses. Recent technological advances in genome editing provide a unique opportunity for the genetic improvement of woody plants. This review examines application of the CRISPR-Cas system to reduce disease susceptibility, alter plant architecture, enhance fruit quality, and improve yields. Examples are discussed of the contemporary CRISPR-Cas system to engineer easily scorable PDS genes, modify lignin, and to alter the flowering onset, fertility, tree architecture and certain biotic stresses.

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DNA-Free Genome Editing via Ribonucleoprotein (RNP) Delivery of CRISPR/Cas in Lettuce

Cited by 37 publications

References 10 publications

Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security

Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience

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