Strategic Design and Engineering of CRISPR/Cas-Powered Sensing Platforms for Enhanced Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Polymerase Chain Reaction (PCR) is the gold standard for nucleic acid detection but requires complex equipment, limiting point-of-care applications.
• CRISPR-Cas systems offer programmable specificity and trans-cleavage activity, revolutionizing biosensing for disease management.

Purpose of the Study:

• To systematically review the evolution of CRISPR-Cas-powered sensing platforms based on signal transduction modalities.
• To analyze different CRISPR-based biosensing architectures and identify challenges and future trends in their clinical translation.

Main Methods:

• Categorization of CRISPR-Cas sensing platforms by signal transduction: fluorescence, naked-eye visual, electrochemical, and electronic/optoelectronic.
• Discussion of biochemical advancements in Cas nucleases (Cas9, Cas12, Cas13) for versatile DNA/RNA targeting.
• Analysis of specific sensing strategies within each modality, including probe design, assay formats, and detection principles.

Main Results:

• CRISPR-Cas platforms demonstrate diverse sensing capabilities, from amplified fluorescence to instrument-free visual and highly sensitive electrochemical/electronic detection.
• Recent discoveries have expanded the range of Cas nucleases and their targeting capabilities for DNA and RNA.
• Significant progress has been made in developing various CRISPR-Dx architectures, including lateral flow assays, colorimetric methods, field-effect transistors, and plasmonic sensors.

Conclusions:

• CRISPR-Cas systems are a disruptive technology for nucleic acid detection, offering alternatives to PCR for point-of-care diagnostics.
• Challenges remain in achieving integrated "sample-to-answer" workflows, high-throughput multiplexing, and digital quantification for clinical adoption.
• Future trends like AI-assisted signal processing and wearable interfaces promise to enhance CRISPR-Dx capabilities and accessibility for precision molecular diagnostics.