ArticlePeerJ2020
A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice.
Article in PeerJ, 2020. 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.
- OsbHLH6 regulates OsMADS57 and amino acid metabolism to impair cold tolerance in rice.Plant cell reports · 2026Article
- Modulating TPP riboswitch activity simultaneously enhances crop yield, nutritional quality and stress tolerance.Nature communications · 2026Article
- TFIIB-Related Protein BRP5/PTF2 Is Required for Both Male and Female Gametogenesis and for Grain Formation in Rice.International journal of molecular sciences · 2023Article
- Comparative Metabolomic Analysis Reveals the Role ofPlants (Basel, Switzerland) · 2023Article
- Article
- LIP5, a MVB biogenesis regulator, is required for rice growth.Frontiers in plant science · 2023Article
- Integrative Analysis of Metabolome and Transcriptome Reveals the Role of Strigolactones in Wounding-Induced Rice Metabolic Re-Programming.Metabolites · 2022Article
- A CRISPR way for accelerating cereal crop improvement: Progress and challenges.Frontiers in genetics · 2022Review
- Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages.Life (Basel, Switzerland) · 2021Review
- A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification.Bio-protocol · 2021Article
- Integrative Metabolomic and Transcriptomic Analyses Reveal Metabolic Changes and Its Molecular Basis in Rice Mutants of the Strigolactone Pathway.Metabolites · 2020Article
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Authors and funding
8 authors.
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Abstract
Rapidly growing genetics and bioinformatics studies provide us with an opportunity to obtain a global view of the genetic basis of traits, but also give a challenge to the function validation of candidate genes. CRISPR/Cas9 is an emerging and efficient tool for genome editing. To construct expression clones for the CRISPR/Cas9, most current methods depend on traditional cloning using Gateway reaction or specific type IIS restriction enzymes and DNA ligation, based on multiple steps of PCR. We developed a system for introducing sgRNA expression cassette(s) directly into plant binary vectors in one step. In this system, one sgRNA expression cassette(s) is generated by an optimized multiplex PCR, in which an overlapping PCR took place. Whilst, two sgRNA expression cassettes were amplified in a single round of PCR. Subsequently, an LR or Golden gate reaction was set up with unpurified PCR product and befitting destination vector. We are able to construct expression clones within 36 h, which greatly improves efficiency and saves cost. Furthermore, the efficiency of this system was verified by an agrobacterium-mediated genetic transformation in rice. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing.
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