ArticlePlant molecular biology2019
Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.
Article in Plant molecular biology, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 32 citations in OpenAlex.
- Molecular understanding of plant pollination, fertilization, seed development and protoplast regeneration.Science China. Life sciences · 2026Review
- Protoplast-Based Functional Genomics and Genome Editing: Progress, Challenges and Applications.Plant, cell & environment · 2026Review
- Isolation and Transfection of Nicotiana benthamiana Mesophyll Protoplasts.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Efficient genome editing in grapevine using CRISPR/LbCas12a system.Molecular horticulture · 2023Article
- Targeted mutagenesis with sequence-specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects.The plant genome · 2023Review
- Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications.Frontiers in plant science · 2023Review
- Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.Plant cell reports · 2022Article
- DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.Plant physiology · 2022Article
- Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts.Animals : an open access journal from MDPI · 2021Article
- Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration.The CRISPR journal · 2021Article
- Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience.Plants (Basel, Switzerland) · 2021Review
- Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.Frontiers in genome editing · 2021Review
- A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement.International journal of molecular sciences · 2020Review
- Multigene CRISPR/Cas9 genome editing of hybrid proline rich proteins (HyPRPs) for sustainable multi-stress tolerance in crops: the review of a promising approach.Physiology and molecular biology of plants : an international journal of functional plant biology · 2020Review
- Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook.International journal of molecular sciences · 2020Review
- How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis.Rice (New York, N.Y.) · 2020Review
- Genome Editing and Protoplast Regeneration to Study Plant-Pathogen Interactions in the Model PlantFrontiers in genome editing · 2020Article
Corrections and comments
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Authors and funding
9 authors at 2 institutions in 2 countries.
Funding
Abstract
key messageProtoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.
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Registered trials
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