ArticleBMC biotechnology2019
Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts.
Article in BMC biotechnology, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers, 1 of them a synthesis that pooled it.
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Who cites it
18 citing papers in PubMed, 1 synthesis or guideline pooled it, 28 citations in OpenAlex.
- Chickpea Biofortification for Cytokinin DehydrogenaseFrontiers in genetics · 2022Pooled it
- Comprehensive benchmarking of genome editing quantification methods for plant applications.iScience · 2025Article
- The Known Unknowns: An Enigmatic Pathway of CCurrent issues in molecular biology · 2025Review
- Genome editing in grain legumes for food security.Frontiers in genome editing · 2025Review
- CRISPR/Cas9 and Agrobacterium tumefaciens virulence proteins synergistically increase efficiency of precise genome editing via homology directed repair in plants.Journal of experimental botany · 2023Article
- Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding.Plants (Basel, Switzerland) · 2023Review
- Genome-wide prediction of activating regulatory elements in rice by combining STARR-seq with FACS.Plant biotechnology journal · 2022Article
- Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity.International journal of molecular sciences · 2022Article
- A roadmap for translational cancer glycoimmunology at single cell resolution.Journal of experimental & clinical cancer research : CR · 2022Review
- Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes forFrontiers in genome editing · 2022Review
- Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.Plants (Basel, Switzerland) · 2021Review
- Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel.Skeletal muscle · 2021Article
- A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example.Plants (Basel, Switzerland) · 2021Article
- Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).Frontiers in genome editing · 2021Article
- First Report of CRISPR/Cas9 Mediated DNA-Free Editing ofInternational journal of molecular sciences · 2021Article
- INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.Nucleic acids research · 2020Review
- Article
- High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato.Scientific reports · 2019Article
Corrections and comments
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Authors and funding
9 authors at 1 institution in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundCRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.
resultsIn this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.
conclusionsFACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.
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