CRISPR/Cas14 provides a promising platform in facile and versatile aptasensing with improved sensitivity

Area of Science:

• CRISPR-Cas systems
• Biosensing technology
• Molecular diagnostics

Background:

• CRISPR technology, particularly Cas12 and Cas13, dominates biosensing.
• The biosensing capabilities of the novel Cas14 enzyme remain underexplored.
• There is a need for versatile and sensitive biosensing platforms.

Purpose of the Study:

• To develop a novel fluorometric biosensor utilizing the Cas14 enzyme.
• To investigate the biosensing potential of Cas14a for various analytes.
• To compare the performance of Cas14a-based biosensors with existing Cas12a systems.

Main Methods:

• Development of HARRY (highly sensitive aptamer-regulated Cas14 R-loop for bioanalysis) biosensor.
• Design of a diblock ssDNA containing Cas14a activator and target-specific aptamer sequences.
• Utilizing Cas14a trans-cleavage activity modulated by target-aptamer binding for fluorescence detection.

Main Results:

• HARRY successfully detected ATP, Cd2+, histamine, aflatoxin B1, and thrombin at low-nanomolar levels.
• The Cas14a-based biosensor exhibited enhanced sensitivity and versatility compared to Cas12a aptasensors.
• Detection limits were found to correlate with aptamer binding affinities, highlighting Cas14a's ssDNA specificity.

Conclusions:

• Cas14a shows significant potential for versatile aptasensing applications.
• The HARRY biosensor represents a promising advancement in CRISPR-based diagnostic tools.
• This work encourages further exploration of Cas14a in the development of next-generation biosensors.