CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a.

Area of Science:

• Molecular Biology
• Biotechnology
• Nucleic Acid Detection

Background:

• CRISPR/Cas13a is a potent RNA detection tool but requires pre-amplification and high temperatures.
• Challenges exist in achieving simple, highly sensitive Cas13a-based RNA detection.
• Understanding Cas13a allosteric regulation by target RNAs is crucial for method development.

Purpose of the Study:

• To investigate the allosteric regulation of Cas13a activation by target RNAs.
• To develop a simplified, highly sensitive RNA detection method using Cas13a.
• To overcome limitations of pre-amplification and elevated temperatures in Cas13a assays.

Main Methods:

• Investigated Cas13a allosteric regulation by target RNA structures and anti-tag sequences.
• Designed a specific CRISPR anti-tag hairpin to modulate Cas13a activity.
• Developed the CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) method.

Main Results:

• Discovered that target RNA secondary structure and anti-tag sequences inhibit Cas13a trans-cleavage.
• Achieved one-step cascade signal amplification for RNA detection without pre-amplification.
• Demonstrated attomolar (10 aM) detection sensitivity for HIV and HCV RNA.
• Validated clinical feasibility using HIV clinical plasma samples.

Conclusions:

• The CARRD method offers a simple, sensitive, and efficient approach for viral RNA detection.
• CARRD operates at room temperature, eliminating the need for pre-amplification.
• The method shows broad applicability for field-deployable diagnostic tools.