CRISPR/Cas13a-Engineered RNA-Based Fluorogenic Biosensor for Label-Free Quantification of RNA in Colorectal Tissues.

Area of Science:

• Biomolecular Engineering
• Molecular Diagnostics
• Cancer Research

Background:

• Colorectal cancer (CRC) poses a significant global health challenge, with early diagnosis remaining difficult.
• Identifying reliable noninvasive biomarkers is crucial for effective CRC detection and management.

Purpose of the Study:

• To develop a sensitive and specific RNA-based biosensor for detecting BRD2 RNA as a biomarker for colorectal cancer.
• To establish a label-free detection method for BRD2 RNA in colorectal tissues using CRISPR/Cas13a technology.

Main Methods:

• Construction of a CRISPR/Cas13a-engineered RNA-based fluorogenic biosensor for BRD2 RNA detection.
• Utilizing Cas13a's trans-cleavage activity and transcription amplification for signal generation.
• Employing Pepper RNA aptamers and a fluorophore (HBC620) for sensitive signal readout.

Main Results:

• The biosensor achieved highly sensitive detection of BRD2 RNA down to 0.39 fM.
• Accurate quantification of BRD2 RNA expression at the single-cell level was demonstrated.
• The biosensor successfully distinguished between colorectal cancer patient tissues and adjacent normal tissues.

Conclusions:

• BRD2 RNA is a promising noninvasive biomarker for colorectal cancer.
• The developed CRISPR/Cas13a-based fluorogenic biosensor provides a sensitive and adaptable platform for early cancer diagnostics.
• The programmability of the biosensor allows for potential adaptation to detect other RNA targets, including noncoding and viral RNAs.