ArticlePlant methods2022
A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants.
Article in Plant methods, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 61 citations in OpenAlex.
- Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.Plant cell reports · 2026Review
- Protoplast-Based Functional Genomics and Genome Editing: Progress, Challenges and Applications.Plant, cell & environment · 2026Review
- Toward Safe and Effective Gene Therapy: Non-Viral Nanostructured Delivery Systems.International journal of nanomedicine · 2026Review
- Tiny but powerful: protoplast isolation inFrontiers in plant science · 2026Article
- Genomic resources and genetic improvement of vital tropical and subtropical fruit crops: current status and prospects.AoB PLANTS · 2025Review
- Somatic Embryogenesis: A Biotechnological Route in the Production of Recombinant Proteins.Biotech (Basel (Switzerland)) · 2025Review
- Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.Plant cell reports · 2025Article
- In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.The Plant journal : for cell and molecular biology · 2025Article
- Directed mutagenesis in fruit crops.3 Biotech · 2025Review
- Development of Protoplast-Based Gene Editing System for Areca Palm.Plants (Basel, Switzerland) · 2025Article
- Form and Function: The Factors That Influence the Efficacy of Nanomaterials for Gene Transfer to Plants.Molecules (Basel, Switzerland) · 2025Review
- DNA delivery into plant tissues using carbon dots made from citric acid and β-alanine.Frontiers in chemistry · 2025Article
- Applications of CRISPR Technologies in Forestry and Molecular Wood Biotechnology.International journal of molecular sciences · 2024Review
- Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome.Plant cell reports · 2024Article
- Creating Climate-Resilient Crops by Increasing Drought, Heat, and Salt Tolerance.Plants (Basel, Switzerland) · 2024Review
- Recent Advances in Tomato Gene Editing.International journal of molecular sciences · 2024Review
- Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome.Frontiers in plant science · 2024Article
- CRISPR/Cas9 ribonucleoprotein mediated DNA-free genome editing in larch.Forestry research · 2024Article
- The importance of genotyping within the climate-smart plant breeding value chain - integrative tools for genetic enhancement programs.Frontiers in plant science · 2024Review
- Generation of transgene-free canker-resistantFrontiers in plant science · 2024Article
Corrections and comments
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Authors and funding
5 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.
resultsHere, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.
conclusionsOur results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.
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