Biochemical plasticity of the <i>Escherichia coli</i><scp>CRISPR</scp> Cascade revealed by <i>in vitro</i> reconstitution of Cascade activities from purified Cas proteins
Bibliographic record
Abstract
The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical association of these proteins, thus producing the Cas5-Cas7 subcomplex that was able to bind specifically to type I-E crRNA with an efficiency comparable to that of the complete Cascade. The crRNA-loaded Cas5-7 was found to bind specifically to the target dsDNA in a PAM-independent manner, albeit with a lower affinity than the complete Cascade, with both spacer sequence complementarity and repeat handles contributing to the DNA targeting specificity. The crRNA-loaded Cas5-7 targeted the complementary dsDNA with detectable formation of R-loops, which was stimulated by the addition of Cas8 and/or Cas11 acting synergistically. Cascade activity reconstitution using purified Cas5-7 and other Cas proteins showed that Cas8 was essential for specific PAM recognition, whereas the addition of Cas11 was required for Cas3 recruitment and target DNA nicking. Thus, although the core Cas5-7 subcomplex is sufficient for specific crRNA binding and basal DNA targeting, both Cas8 and Cas11 make unique contributions to efficient target recognition and cleavage.
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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 | 0.000 |
| 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".