Multicolor Amplification-Free RNA Detection with Cas13a and Cas13b

Area of Science:

• Molecular Diagnostics
• CRISPR Technology
• Infectious Disease Surveillance

Background:

• The COVID-19 pandemic highlights the need for rapid, accurate multiplex diagnostics.
• Current multiplex nucleic acid amplification tests (NAATs) face challenges with amplification bias and long turnaround times.
• Faster, reliable alternatives are crucial for timely pathogen identification.

Purpose of the Study:

• To develop an amplification-free, single-molecule genetic test for simultaneous detection of dual RNA targets.
• To introduce multicolor SATORI (mSATORI) utilizing CRISPR-Cas13a and Cas13b.
• To assess the diagnostic performance of mSATORI for infectious disease detection.

Main Methods:

• Developed multicolor SATORI (mSATORI), an amplification-free, single-molecule genetic test.
• Leveraged complementary activities of CRISPR-Cas13a and Cas13b for simultaneous dual RNA target detection.
• Validated mSATORI using clinical specimens for Influenza A and SARS-CoV-2 RNA detection.

Main Results:

• mSATORI identified Influenza A and SARS-CoV-2 RNAs in approximately 10 minutes.
• Achieved analytical limits of detection (LoD) of 86 aM for Influenza A and 52 aM for SARS-CoV-2.
• Demonstrated femtomolar LoD in clinical specimens (550 aM for Influenza A, 640 aM for SARS-CoV-2) with >80% sensitivity and 100% specificity.

Conclusions:

• mSATORI is a promising platform for next-generation molecular diagnostics.
• The test offers rapid, accurate, and sensitive detection of viral RNA.
• mSATORI has broad implications for clinical implementation, outbreak preparedness, and global surveillance.