ArticlePlant methods2024
An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat.
Article in Plant methods, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed.
- Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.Plant cell reports · 2026Review
- Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.Journal of integrative plant biology · 2026Article
- Golden Promise-rapid, a fast-cycling and transformable barley genotype.Journal of experimental botany · 2026Article
- Wheat's Up with CRISPR-Cas-Current Advances, Obstacles and Perspectives.International journal of molecular sciences · 2026Review
- Arabidopsis OST1 homologs of barley are involved in stomatal regulation.Journal of experimental botany · 2026Article
- Advances in CRISPR/Cas systems for engineering abiotic stress tolerance in plants: mechanisms and future prospects.Planta · 2026Review
- Reduced Cas9 transgene silencing by incorporation of intron sequences.Nature communications · 2025Article
- Efficient genome editing by CRISPR-Cas12a in common wheat via shoot apical meristem delivery.Plant physiology · 2025Article
- Improvement of simultaneous genome editing of homoeologous loci in polyploid wheat using CRISPR/Cas9 applying tRNA processing system.Plant biotechnology (Tokyo, Japan) · 2025Article
- Isolation and transformation of perennial ryegrass (Frontiers in plant science · 2025Article
- Recent advances of CRISPR-based genome editing for enhancing staple crops.Frontiers in plant science · 2024Review
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7 authors.
Funding
Abstract
backgroundCRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.
resultsWe identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.
conclusionBased on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.
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