CRISPR-based Diagnostics
Related: CRISPR-Cas in Bacteria · Isothermal NAAT · qPCR · PCR
1. Principle
CRISPR diagnostic assays use programmed Cas enzymes (Cas12, Cas13, etc.) that, after recognizing a target sequence, unleash collateral cleavage of reporter molecules — producing a fluorescent or lateral-flow signal with single-nucleotide discrimination potential.
2. Step-by-Step Procedure (conceptual)
- Extract NA (or use crude lysis).
- Pre-amplify target (Isothermal NAAT or PCR) — often required for clinical sensitivity.
- Apply CRISPR–guide RNP programmed to pathogen/resistance motif.
- Collateral cleavage activates reporter → read on fluorimeter or paper strip.
- Interpret with controls.
3. Interpretation
- High specificity from guide–target complementarity + PAM constraints.
- Pitfalls: Still needs validation like any NAAT; pre-amplification reintroduces contamination risks; multiplexing maturing.
4. Clinical Use Cases
- Emerging rapid tests for viruses and resistance alleles (platform-dependent availability).
- Field / low-resource adaptations (paper readouts).
- Research companion to AI Diagnostics in Microbiology and synthetic biology.
5. Comparison with Other Methods
| Method | Recognition | Hardware |
|---|---|---|
| CRISPR dx | Guide RNA + Cas | Simple to moderate |
| qPCR | Primers/probes | Thermocycler |
| Isothermal NAAT | Primers ± CRISPR | Heater / cartridge |
6. Mnemonic / Visual Aid
Find the match → cut the reporter — collateral cleavage is the alarm bell.
Active Recall
- Cas12 vs Cas13 — DNA or RNA targeting preference?
- Why is pre-amplification still common?
- How does this relate to adaptive bacterial immunity?