Programmable RNA detection with CRISPR-Cas12a.

Area of Science:

• Biotechnology
• Molecular Biology
• Genomics

Background:

• CRISPR-Cas12a is a key bioengineering tool for DNA cleavage in diagnostics.
• Existing methods require reverse transcription or strand displacement for RNA detection.

Approach:

• Developed SAHARA (Split Activators for Highly Accessible RNA Analysis) for direct RNA detection using Cas12a.
• Exploited Cas12a's ability to recognize RNA at the PAM-distal region of crRNA.
• Utilized a PAM-proximal DNA as a switch to control Cas12a trans-cleavage activity.

Key Points:

• SAHARA enables amplification-free detection of RNA, including miRNA-155 and hepatitis C virus RNA, at picomolar concentrations.
• The method is Mg2+ concentration- and pH-dependent, operates robustly at room temperature, and shows improved specificity.
• SAHARA supports multiplexed detection of distinct DNA and RNA targets using pooled CRISPR-Cas12a complexes.

Conclusions:

• SAHARA is a versatile and powerful nucleic acid detection platform.
• The platform offers a simple, sensitive, and specific approach for RNA analysis.
• SAHARA has potential for expansion to other CRISPR-Cas enzymes and multiplexed applications.