A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440
Bibliographic record
Abstract
Abstract Background Pseudomonas putida KT2440, a non-pathogenic soil bacterium, is a key platform strain in synthetic biology and industrial applications due to its robustness and metabolic versatility. Various systems have been developed for genome editing in P. putida, including transposon modules, integrative plasmids, recombineering systems, and CRISPR/Cas systems. However, rapid iterative genome editing is limited by complex and lengthy processes. Results We discovered that the pBBR1MCS2 plasmid carrying the CRISPR/Cas9 module could be easily cured in P. putida KT2440 at 30 oC. We then developed an all-in-one CRISPR/Cas9 system for yqhD and ech-vdh-fcs deletions, respectively, and further optimized the editing efficiency by varying homology arm lengths and target sites. Sequential gene deletions of vdh and vanAB were carried out rapidly using single-round processing and easy plasmid curing. This system’s user-friendliness was validated by 3 researchers from two labs for 9 deletions, 3 substitutions, and 2 insertions. Finally, iterative genome editing was used to engineer P. putida for valencene biosynthesis, achieving a 10-fold increase in yield. Conclusions We developed and applied a rapid all-in-one plasmid CRISPR/Cas9 system for genome editing in P. putida. This system requires less than 1.5 days for one edit due to simplified plasmid construction, electroporation and curing processes, thus accelerating the cycle of genome editing. To our knowledge, this is the fastest iterative genome editing system for P. putida. Using this system, we rapidly engineered P. putida for valencene biosynthesis for the first time, showcasing the system’s potential for expanding biotechnological applications.
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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 | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.065 | 0.004 |
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; both teacher heads agree on what is shown here.
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".