Introduction

As infectious diseases spread faster than ever, the ability to accurately distinguish between multiple viruses and their variants in a single test is critical. Researchers at KAIST and an international team have developed a groundbreaking diagnostic technology that does just that: it uses the 'speed' of CRISPR gene scissors to simultaneously identify different pathogens. This guide breaks down the process into clear, actionable steps, so you can understand how this method works and apply it in your lab or diagnostic setting.

What You Need

• CRISPR-Cas12 or Cas13 system (enzyme and buffer)
• Guide RNAs designed for each target virus or variant
• Target nucleic acid samples (RNA or DNA)
• Fluorescent reporters or other detection molecules
• Real-time fluorescence reader (e.g., plate reader)
• Computer software for analyzing reaction speed curves
• Pipettes and sterile tubes
• Temperature control (incubator or thermocycler)

Step-by-Step Instructions

1. Step 1: Design guide RNAs specific to each target
   For every virus or variant you want to detect, create a unique guide RNA that binds only to its genetic sequence. This ensures your CRISPR scissors only cut when the correct target is present.
2. Step 2: Prepare the CRISPR reaction mix
   Combine the Cas12 or Cas13 enzyme with its buffer, the set of guide RNAs, and fluorescent reporters in a single tube. Mix gently and keep on ice.
3. Step 3: Add your sample
   Pipette the extracted nucleic acid sample into the reaction mix. This initiates the CRISPR machinery – if the target sequence is present, the Cas enzyme will bind and cleave the reporter, producing fluorescence.
4. Step 4: Monitor fluorescence in real time
   Place the reaction tube into a real-time fluorescence reader. Record the fluorescence intensity every 30 seconds for 30–60 minutes. The speed at which fluorescence increases is directly related to the amount and type of target.
5. Step 5: Analyze speed patterns
   Use software to plot fluorescence over time for each reaction. Different viruses and variants produce characteristic speed curves – for example, a rapid rise might indicate a high viral load or a specific variant. By comparing the shape and slope to known standards, you can identify which pathogens are present and even their relative quantities.
6. Step 6: Decode multiple targets simultaneously
   Because you included several guide RNAs in one reaction, the overall speed pattern is a composite of all active CRISPR systems. The new KAIST technology uses advanced algorithms to deconvolve this mixed pattern, revealing each virus or variant individually – all in a single well.
7. Step 7: Validate with positive and negative controls
   Always run a sample without any target (negative control) and samples with known virus sequences (positive controls). This ensures your speed patterns are accurate and not due to contamination or non-specific cleavage.
8. Step 8: Repeat for different sample types
   The method works with both RNA viruses (like SARS-CoV-2) and DNA viruses (like hepatitis B). Adjust the CRISPR system accordingly (Cas13 for RNA, Cas12 for DNA).

Tips for Success

• Optimize guide RNA concentration – too little may cause slow signals, too much increases background.
• Maintain strict temperature control; the CRISPR reaction speed is temperature-sensitive (optimal around 37°C).
• Use high-quality fluorescent reporters to avoid bleaching or low signal-to-noise.
• Test each guide RNA individually first to confirm it produces a unique speed pattern before mixing.
• Regularly calibrate your real-time reader for consistent results.
• For clinical samples, pre-process to remove inhibitors that could slow down the CRISPR reaction.
• Store all reagents at recommended temperatures to maintain enzyme activity.

This approach, pioneered by KAIST, transforms a single CRISPR reaction into a powerful multiplex diagnostic tool. By focusing on the speed of fluorescence generation rather than just its intensity, you can identify multiple viruses and their variants simultaneously – saving time, cost, and sample volume.