Strong expression of Cas9 under a new 3′-truncated TEF1α promoter enhances genome editing in Yarrowia lipolytica
Bibliographic record
Abstract
The non-conventional yeast Yarrowia lipolytica is gaining interest in biotechnology as a workhorse for the production of proteins, lipids and other biomolecules. Site-specific genome editing is however limited in this yeast. This was much improved by the recent adaptation of a CRISPR-Cas9 genome editing protocol for Y. lipolytica based on a tRNA-sgRNA fusion. Nonetheless, in the latter protocol, Cas9 is under the control of a synthetic hybrid promoter, pUAS1B8-TEF(136) that although reported as strong, yet its tandem repeats might be associated with in vivo and in vitro inconveniences like polymerase slippage, random genetic rearrangements and cloning difficulties. Here we report an optimized synthetic TEF promoter to drive Cas9 expression, pTEF(-41-406)-Kozak, which is a rationally 3’-truncated version of the already known 5’-truncated pTEF(406) promoter of Y. lipolytica fused to a synthetic Kozak sequence. Our comparison of the promoters’ strength using hrGFP reporters and RT-qPCR revealed that the synthetic pTEF(-41-406)-Kozak maintains the same expression strength as that of pTEF(406), which is at least 5-folds higher than that of synthetic pUAS1B8-TEF(136). This pTEF(-41-406)-mediated high expression of Cas9 was not associated with any growth defects. Moreover, Cas9 under pTEF(-41-406)-Kozak increased genome editing efficiencies by 40 % relative to that under pUAS1B8-TEF(136), and this was further concordant by observation of rates of phenotypic losses resulting from pTEF(-41-406)-Kozak-Cas9-mediated gene deletions. This is the first study conducting rational 3’-truncation in TEF promoter in Y. lipolytica based on in silico analysis of promoter sequence and structure, which can be extended to the engineering of other yeast promoters to generate small-sized synthetic biology parts for convenient engineering of biological systems. Here, we provide a strong Cas9 expression cassette for a more convenient and efficient CRISPR-Cas9-mediated genome editing in Y. lipolytica which will allow harnessing the full potential of this industrial strain.
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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) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 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".