Coupling CRISPR/Cas9 and Lambda Red Recombineering System for Genome Editing of Salmonella Gallinarum and the Effect of ssaU Knock-Out Mutant on the Virulence of Bacteria
Bibliographic record
Abstract
The poultry industry in developing countries still faces a significant threat from fowl typhoid, a disease caused by Salmonella Gallinarum that has been well contained in more economically developed countries. In addition to the virulence exhibited by large virulence plasmid (85 kb), Salmonella Pathogenicity Island 2 in S. Gallinarum plays a key role in mediating disease through its type III secretion systems (TTSS). The TTSS secrete effector protein across the Salmonella containing vacuoles and mediate the internalization of bacteria by modulating vesicular passage. In this study, candidate virulent ssaU gene (~1 kb) encoding type III secretion system was successfully deleted from indigenously isolated S. Gallinarum genome through homology-directed repair using CRISPR/Cas9 and lambda recombination systems. CRISPR/Cas9-based genome editing of poultry-derived Salmonella Gallinarum has not been previously reported, which might be linked to a lack of efficiency in its genetic tools. This is the first study which demonstrates a complete CRISPR/Cas9-based gene deletion from this bacterial genome. More importantly, a poultry experimental model was employed to assess the virulence potential of this mutant strain (ΔssaU_SG18) which was unable to produce any mortality in the experimentally challenged birds as compared to the wild type strain. No effect on weight gain was observed whereas bacteria were unable to colonize the intestine and liver in our challenge model. This in vivo loss of virulence in mutant strain provides an excellent functionality of this system to be useful in live vaccine development against this resistant and patho genic bacteria.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 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 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 teacher head, 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".