Expanding the CRISPR toolbox by engineering Cas12a orthologs of metagenomic discovery
Bibliographic record
Abstract
Abstract Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility in genome editing and molecular diagnostic applications. However, the widespread adoption of CRISPR-Cas12a nucleases and their variants has been hindered by the requirement for a specific protospacer adjacent motif (PAM), relatively low CRISPR RNA (crRNA) activity and the inability to multiplex nucleic acid detection alone. To overcome these limitations, we employed a comprehensive framework combined with AlphaFold2 to de novo mine 1,261 previously unexploited Cas12a orthologs from the global microbiome. Following experimental analysis, we identified the most promising 21 Cas12a nuclease orthologs and designated them “Genie scissor 12” (Gs12). Our analysis uncovered two exceptional variants among these newly identified orthologs: Gs12-10, a first natural PAM-less Cas12a ortholog, which can recognize 52 distinct PAM types, representing a significant 1.8-fold expansion in recognition range compared to the relative LbCas12a PAM; and Gs12-7MAX, an engineered variant of Gs12-7 that exhibited 1.27-fold higher editing efficiency than enAsCas12a-HF. Furthermore, we harnessed Gs12-1, Gs12-4, Gs12-9, and Gs12-18, along with their corresponding engineered crRNAs, to develop a powerful four-channel multiplexed CRISPR-based nucleic acid detection system. The discovery of diverse functions in Cas12a offers a deeper understanding of the CRISPR/Cas12a family. Also, it holds great promise for expanding its applications and uncovering the untapped potential of other CRISPR/Cas systems. Graphical Abstract
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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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| 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".