ArticlePlant cell reports2016
Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.
Article in Plant cell reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 96 papers.
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Who cites it
96 citing papers in PubMed, 229 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
- Transgene-free genome editing in plants.aBIOTECH · 2026Review
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- CRISPR-Cas technologies for precision genome editing in plants: advances, applications, and future perspectives.Frontiers in plant science · 2026Review
- CRISPR/Cas9-Targeted Gene Editing of Allergenic Profilin-EncodingPlants (Basel, Switzerland) · 2025Article
- A novel protocol for protoplast isolation, transfection, and culture in Cannabis sativa L.BMC plant biology · 2025Article
- Emerging trends in transgene-free crop development: insights into genome editing and its regulatory overview.Plant molecular biology · 2025Review
- Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.Transgenic research · 2025Article
- Advancements in plant transformation: from traditional methods to cutting-edge techniques and emerging model species.Plant cell reports · 2024Review
- How to use CRISPR/Cas9 in plants: from target site selection to DNA repair.Journal of experimental botany · 2024Review
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- CRISPR/Cas genome editing in plants: mechanisms, applications, and overcoming bottlenecks.Functional & integrative genomics · 2024Review
- Editing of the ethylene biosynthesis gene in carnation using CRISPR-Cas9 ribonucleoprotein complex.Plant methods · 2024Article
- CRISPR/Cas9 ribonucleoprotein mediated DNA-free genome editing in larch.Forestry research · 2024Article
- Generation of transgene-free canker-resistantFrontiers in plant science · 2024Article
- Putting CRISPR-Cas system in action: a golden window for efficient and precise genome editing for crop improvement.GM crops & food · 2023Review
- 3Bs of CRISPR-Cas mediated genome editing in plants: exploring the basics, bioinformatics and biosafety landscape.Physiology and molecular biology of plants : an international journal of functional plant biology · 2023Review
- Improvements in Protoplast Isolation Protocol and Regeneration of Different Cabbage (Plants (Basel, Switzerland) · 2023Article
- Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation.Nature communications · 2023Article
- Review
36 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
key messageSite-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.