ArticleBMC biotechnology2019
gRNA validation for wheat genome editing with the CRISPR-Cas9 system.
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 33 papers.
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Who cites it
33 citing papers in PubMed, 78 citations in OpenAlex.
- Engineering Wheat for Compound Climate Extremes: Molecular Trade-Offs, Stress Memory, and Precision Genome Editing for Yield-Stable Resilience.Plants (Basel, Switzerland) · 2026Review
- Efficient in-vitro regeneration and transformation for CRISPR/Cas9-mediated genome editing of phytoene desaturase (PDS) gene in pea (Pisum sativum L.).Plant cell reports · 2026Article
- Review
- CRISPR/Cas-mediated genome editing: playing a versatile role in mitigating the challenges of sustainable rice improvement.3 Biotech · 2025Review
- Review
- The extent of multiallelic, co-editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield.Plant biotechnology journal · 2024Article
- Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity.bioRxiv : the preprint server for biology · 2024Article
- Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.Nature plants · 2024Article
- Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane.Frontiers in genome editing · 2024Article
- Genome Editing to Produce Knockout Mutations of Seed Dormancy Genes in Wheat.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Alien introgression to wheat for food security: functional and nutritional quality for novel products under climate change.Frontiers in nutrition · 2024Review
- Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.Scientific reports · 2023Article
- Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.International journal of molecular sciences · 2023Review
- Recommendations for the Assessment of Potential Environmental Effects of Genome-Editing Applications in Plants in the EU.Plants (Basel, Switzerland) · 2023Review
- Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures.International journal of molecular sciences · 2023Article
- Fine-tuning CRISPR/Cas9 gene editing in common bean (Frontiers in plant science · 2023Article
- CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.Planta · 2022Article
- CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses.Current issues in molecular biology · 2022Review
- Application of CRISPR/Cas9 System for Efficient Gene Editing in Peanut.Plants (Basel, Switzerland) · 2022Article
- Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants.ACS agricultural science & technology · 2022Review
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.
resultsThe seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.
conclusionsWe have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.
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