Development of a Csy4-Processing TRV-Based CRISPR/Cas9 Genome Editing System in Nicotiana benthamiana
Bibliographic record
Abstract
Abstract The CRISPR/Cas9 system is a site-specific genome editing tool that has been widely used in various plant species. The plant virus-based gRNA (guide RNA) delivery system, which differs from the typical Agrobacterium-mediated transformation, is an attractive method to facilitate the application of CRISPR/Cas9. The virally delivered gRNA is usually driven by heterologous plant U6 or viral promoters (e.g., pea early-browning virus, PEBV; barley stripe mosaic virus, BSMV). However, heterologous promoters may have poor performance in some cases. In this paper, a feasible option to detach gRNA(s) from the virus genome is employed. Specifically, the Csy4-RNA processing system is used to release gRNA(s) from the tobacco rattle virus (TRV). The coding sequences of Cas9 and Csy4 nucleases are cloned into a single polycistronic expression cassette under an estrogen-inducible promoter in a binary vector, and the gRNA is cloned into the TRV genome flanked by two 20 bp Csy4 recognition sites. The results show that the Csy4-processing TRV-based delivery system works effectively in targeting single and multiple sites, nucleotide replacement, and large fragment deletion in Cas9-mediated genome editing via transient expression in Nicotiana benthamiana. The Csy4-TRV is a promising gRNA(s) processing and delivery system for CRISPR/Cas9 genome editing. This method can be easily adapted to other plant RNA viruses, facilitating the application of the CRISPR/Cas9 system in plants.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".