An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing

Area of Science:

• Molecular Biology
• Biotechnology
• Nucleic Acid Chemistry

Background:

• CRISPR-Cas systems offer precise nucleic acid recognition and cleavage, valuable for biosensing.
• Current CRISPR-based biosensing methods face challenges in complexity, efficiency, and probe design.

Purpose of the Study:

• To develop a novel CRISPR-Cas based approach for highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA.
• To overcome limitations of existing methods by integrating Cas protein functions through an autocatalysis mechanism.

Main Methods:

• Developed CRISPR-Cas Autocatalysis Amplification (CALSA) using LNA-modified Split Activators and the LbCas12a system.
• Constructed an autocatalysis-driven positive feedback loop with split ssDNA activators and site-directed trans-cleavage.
• Utilized LNA modifications to mediate trans-cleavage for enhanced signal amplification.

Main Results:

• CALSA enabled one-pot, real-time detection of genomic DNA and cell-free DNA (cfDNA) from tumor cell lines.
• Achieved high sensitivity and single-base specificity in DNA detection.
• Demonstrated remarkably short reaction times, showcasing efficient cascade signal amplification.

Conclusions:

• CALSA offers a powerful and programmable tool for cascade signal amplification in nucleic acid detection.
• The high sensitivity and specificity of CALSA highlight its potential for advanced biosensing and diagnostic applications.
• CALSA opens new avenues for future clinical applications in diagnostics and molecular detection.