Amplification-free nucleic acids detection with next-generation CRISPR/dx systems.

Area of Science:

• Molecular Diagnostics
• Biotechnology
• Biosensing

Background:

• CRISPR-based diagnostics (CRISPR/Dx) are transforming molecular diagnostics, enabling point-of-care testing (POCT).
• Sensitive detection of low-abundance nucleic acids often necessitates preamplification, limiting current CRISPR/Dx applications.
• Amplification-free CRISPR/Dx systems have emerged to overcome this limitation, enhancing signal detection sensitivity.

Purpose of the Study:

• To critically review and categorize recent advances in amplification-free CRISPR/Dx systems for nucleic acid detection.
• To discuss the various signal enhancement strategies employed in these systems.
• To identify the pitfalls and future perspectives of amplification-free CRISPR/Dx technologies.

Main Methods:

• Classification of amplification-free CRISPR/Dx systems into five groups based on signal enhancement strategies.
• Integration of CRISPR/Cas12a and/or CRISPR/Cas13a with other catalytic enzymes, oligonucleotides, nanomaterials, biosensors, or advanced detection platforms.
• Critical discussion of the advantages, limitations, and future directions for each category.

Main Results:

• Five main categories of amplification-free CRISPR/Dx systems were identified based on signal enhancement approaches.
• These systems leverage diverse components like enzymes, oligonucleotides, nanomaterials, and advanced biosensors for improved detection.
• The reviewed systems demonstrate potential for sensitive nucleic acid detection without target amplification.

Conclusions:

• Amplification-free CRISPR/Dx systems offer a promising avenue for rapid, cost-effective, and ultrasensitive on-site nucleic acid detection.
• Continued refinement of these systems is crucial for establishing CRISPR/Dx as a leading diagnostic technology.
• These advancements pave the way for widespread adoption of CRISPR/Dx in various diagnostic settings.