Efficient in vitro and in vivo self-repression of SpCas9 gene using a molecular Hara-Kiri method.
Bibliographic record
Abstract
Abstract The CRISPR/Cas9 system is currently a major revolution in the field of biology. Because of its simplicity compared to other endonucleases, this system is being experimented in diverse fields. However, a major disadvantage is the toxicity linked to sustained Cas9 expression. In the present study, we present an approach to effectively suppress the expression of the Streptococcus pyogenes Cas9 (SpCas9) gene. This approach that we call the molecular Hara-Kiri method, involves two sgRNAs targeting two sequences in the SpCas9 gene. The SpCas9 enzyme binds to the Protospacer Adjacent Motifs following the two sequences targeted by the sgRNAs and induces two Double Strand Breaks (DSBs) in its own gene (Hara-Kiri). The sequence located between the DSBs is then deleted. Most of the time, the SpCas9 gene is repaired by Non-Homologous End Joining without INDELs. By adequately selecting the targeted sequences, the junction of the SpCas9 gene residues generates a TAA type stop codon within this truncated gene to effectively suppress its expression. This results in dramatic decrease of the SpCas9 protein in vitro and in vivo.
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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) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 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".