Crispr_C
DNA Plasmids
Circular Vectors
Terminology
- Virus-Induced Genome Editing VIGE
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- Floral Dip Technique
- "However, transgenes derived from the AMGT vector are randomly integrated into the host genome; this might result in the disruption of host gene expression. Such limitations of AMGT call for alternative physical or chemical methods to expand the scope of plant engineering."
Overview
- CRISPR-based gene editing in plants: Focus on reagents and their delivery tools Ebrahimi et al. 2024 Review Article
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Table 1: Summary of CRISPR delivery methods in plant genome editing
| Method | Description | Applications and Advantages | Limitations and Challenges |
|---|---|---|---|
| Agrobacterium-Mediated Transformation (AMT) | Agrobacterium tumefaciens delivers CRISPR components through T-DNA transfer, allowing gene editing in various plants. | - AMT effectively modifies wheat, tomato, tobacco, etc. - CRISPR-Cas9 via AMT enhances grain weight, yields, and provides resistance to powdery mildew. | - Limited cargo capacity. - Off-target effects can occur. |
| Biolistic Transformation | Particles coated with CRISPR components are bombarded into plant cells, introducing DNA without direct cell contact. | - Utilized in maize, wheat, barley, rice, etc. - Overcomes limitations of other methods in introducing foreign DNA into plant tissues. | - Tissue culture is often required for regeneration. - Limited efficiency compared to other methods. |
| Electroporation | Electrical current creates temporary pores in cell membranes, facilitating efficient entry of CRISPR components. | - High efficiency and minimal off-target effects. - Used in tobacco and switchgrass for CRISPR-Cas9 delivery. | - Dependency on electric field application. |
| Expression of Developmental Regulators | WUS and BBM induce new embryogenesis, transforming rejected lines. Ectopic expression of regulators (IPT, STM, BBM, GRF4, GIF1) enhances transformation frequencies. | - Successful transformation of previously rejected lines. - Increased genome-edited plant numbers with CRISPR-Cas9 and developmental regulators. | - Poor performance in DNA delivery and plant regeneration. |
| RNA Viruses and Mobile Guide RNAs | TRV, a positive-strand RNA virus, delivers sgRNAs into Cas9-overexpressing plants through Agrobacterium infiltration. Sonchus yellow net rhabdovirus delivers sgRNA and SpCas9. | - TRV achieves systemic, heritable gene editing. - Sonchus yellow net rhabdovirus induces heritable mutations. | - Low cargo capacity of viruses. - TRV can't transmit to progeny. |
CRISPR-based gene editing in plants: Focus on reagents and their delivery tools
Virus-Induced Genome Editing
Research Articles
- High-efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus Nagalakshmi et al. 2022 Research Article
- "[First], we tested editing in Arabidopsis using TRV with sgRNA targeted to PHYTOENE DESATURASE3 (AtPDS3) fused to tRNAIleu."
- "Agrobacterium harboring TRV1 and TRV2::sgRNAAtPDS3-tRNAIleu were delivered into Col-0 expressing SpCas9 (Col-0::SpCas9) by syringe infiltration of leaves, agro-pricking, and agro-flooding meth ods (see Supplemental Text S1 for details) (Supplemental Figure S2)."
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- "About 3% (1/36) and 8% (3/36) of the plants showed photobleached regions on the systemic leaves with leaf infiltration and agro-pricking methods, respectively (Supplemental Figure S3A). With agro-flooding, about 22% of the plants (8/36) showed the photobleaching phenotype (Supplemental Figure S3B) indicating that the agro-flooding is more efficient for sgRNA delivery into Arabidopsis."
- "[Second], we evaluated the movement of TRV with sgRNAAtPDS3 fused to mAtFT and tRNAMet (Supplemental Figure S1) in Arabidopsis using agro-flooding method."
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- "A substantially higher amount of virus was detected in systemic leaves of TRV2::sgRNAAtPDS3-tRNAIleu infected plants compared with sgRNAAtPDS3 fused to mAtFT, tRNAMet, or sgRNAAtPDS3 alone (Figure 1A) indicating that the tRNAIleu facilitates better TRV movement in Arabidopsis."
- These results indicate that TRV2::sgRNAAtPDS3-tRNAIleu induces high-efficiency somatic and heritable editing in Arabidopsis.
- Heritable virus-induced germline editing in tomato Oh et al. 2025 Research Article
- "These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using [virus-induced germline editing]."
- Virus-mediated, heritable gene editing in groundcherry (Physalis grisea) Tibebu et al. 2026 Research Article
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Methodology
- Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two Ordon et al. 2023 Methodology
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- Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System Mahas et al. 2019 Methodology
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Floral Dip Technique
Research Articles
- CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family Angulo et al. 2023 Research Article
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- Direct protein delivery into intact Arabidopsis cells for genome engineering Furuhata et al. 2024 Research Article
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Methodology
- Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana Clough et al. 1998 Methodology
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- Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana Davis et al. 2009 Methodology
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- An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol Logemann et al. 2006 Methodology
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Review Articles
Good for background information and broad concepts.
Review Articles
- Arabidopsis thaliana Floral Dip Transformation Method Bent et al. 2006 Review Article
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CRISPR/Cas9
Research Articles
- Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula Wolabu et al. 2020 Research Article
- "The improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both [Arabidopsis and M. truncatula]."