CRISPR-Cas13 Based Point-of-Care Diagnostics for Rapid Detection of Antimicrobial Resistance Genes
Abstract
Antimicrobial resistance (AMR) poses a critical global health threat, yet conventional culture-based susceptibility testing requires 48-72 hours. We developed a CRISPR-Cas13a lateral flow assay (CRISPR-POC) that detects five clinically relevant resistance genes (blaKPC, blaNDM, mecA, vanA, mcr-1) directly from clinical specimens within 45 minutes. The platform combines isothermal RPA pre-amplification with Cas13a collateral cleavage of FAM-BHQ-labeled reporters, producing visible test-line signals on lateral flow strips. Clinical validation on 312 urine and wound swab samples achieved 96.8% sensitivity and 98.2% specificity compared to whole-genome sequencing, with a limit of detection of 50 copies/reaction. The assay operates at room temperature after a single 37°C incubation, making it suitable for resource-limited settings and emergency departments.
Keywords: CRISPR-Cas13, antimicrobial resistance, point-of-care diagnostics, lateral flow assay, rapid detection
1. Introduction
The World Health Organization has identified antimicrobial resistance as one of the top ten global public health threats, with carbapenem-resistant Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) causing over 700,000 deaths annually. Rapid identification of resistance determinants at the point of care is essential for guiding appropriate antibiotic therapy and implementing infection control measures.
CRISPR-Cas13 systems offer unique advantages for nucleic acid detection through programmable RNA-guided collateral cleavage activity. Unlike Cas9-based detectors, Cas13a exhibits robust trans-cleavage of single-stranded RNA reporters upon target recognition, enabling signal amplification without additional enzymatic steps. When integrated with lateral flow readout, this mechanism provides a visually interpretable result suitable for non-specialist operators.
2. Assay Design and Optimization
The CRISPR-POC assay workflow comprises three steps: (1) rapid lysis of bacterial cells in clinical specimens using a chaotropic buffer, (2) recombinase polymerase amplification (RPA) at 37°C for 20 minutes to amplify target gene fragments, and (3) Cas13a detection with lateral flow readout for 15 minutes. crRNAs were designed to target conserved regions of blaKPC, blaNDM, mecA, vanA, and mcr-1 with ≤2 mismatches tolerance.
Table 1. Optimized reaction conditions for CRISPR-POC detection of AMR gene targets
| Component | Concentration | Function | Incubation |
|---|---|---|---|
| RPA primers | 480 nM each | Isothermal amplification | 37°C, 20 min |
| LwaCas13a | 100 nM | Target recognition & cleavage | 37°C, 15 min |
| crRNA | 120 nM | Target specificity | — |
| FAM-BHQ reporter | 500 nM | Signal generation | — |
| MgCl₂ (RPA) | 14 mM | Enzyme activation | — |
3. Analytical and Clinical Performance
Analytical sensitivity was determined using serial dilutions of synthetic gene targets and cultured bacterial isolates. All five targets were detected at 50 copies/reaction with no cross-reactivity among the panel. Clinical validation was performed on 312 prospectively collected specimens from three hospitals, comparing CRISPR-POC results against Illumina whole-genome sequencing as the reference standard.
Table 2. Confusion matrix summary for CRISPR-POC clinical validation across all five gene targets
| Metric | Value | 95% CI |
|---|---|---|
| Overall sensitivity | 96.8% | 94.1–98.4% |
| Overall specificity | 98.2% | 96.5–99.2% |
| Positive predictive value | 95.4% | 92.3–97.5% |
| Negative predictive value | 98.7% | 97.1–99.5% |
| Limit of detection | 50 copies/reaction | — |
4. Conclusions
The CRISPR-POC platform enables rapid, accurate detection of five major AMR genes directly from clinical specimens in under 45 minutes. Its room-temperature-compatible lateral flow readout and minimal equipment requirements make it deployable in emergency departments, outpatient clinics, and low-resource settings. Future work will expand the gene panel to include ESBL and colistin resistance markers and integrate multiplex crRNA arrays for simultaneous detection of all targets on a single strip.
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This article is published under the Creative Commons Attribution 4.0 International License (CC BY 4.0).