From Self-Processing to Responsive Assembly Enabling an Autocatalytic Cas13a Circuit for Enhanced Biosensing.

Area of Science:

• Molecular Biology
• Biotechnology
• CRISPR Technology

Background:

• CRISPR-Cas13a systems face challenges in biosensing due to crRNA design limitations.
• Effective activation of Cas13a trans-cleavage requires specific strategies, especially concerning the crRNA seed region.

Purpose of the Study:

• To develop an enhanced CRISPR-Cas13a biosensing system with improved sensitivity and specificity.
• To address the limitations of current Cas13a systems, including off-target cleavage and detection speed.
• To enable rapid, point-of-care diagnostics for various targets like miRNA, mRNA, and viral DNA.

Main Methods:

• Developed an autocatalytic Cas13a circuit (PRA-Cas13a) utilizing engineered pre-crRNA as a molecular switch.
• Integrated a dual-UUU site DNA switch template and T7 RNA polymerase for signal amplification, creating a "processing-assembly-amplification" cycle.
• Validated the system using diverse targets (miRNA, mRNA, viral DNA) and clinical samples (cell lines, cervical swabs).

Main Results:

• Achieved attomolar (aM) detection limits with visual field detection in 10 minutes using lateral flow test strips.
• Demonstrated significantly superior sensitivity compared to conventional CRISPR methods, even with single-base mutations.
• Successfully detected survivin mRNA in cell lines and HPV16 in clinical samples with high concordance to qPCR.

Conclusions:

• The PRA-Cas13a strategy effectively leverages self-processing assembly and autocatalytic signal amplification for enhanced biosensing.
• The system overcomes off-target cleavage issues common in Cas13a systems and expands target applicability.
• PRA-Cas13a shows high specificity and potential for point-of-care diagnostics in cancer and viral disease detection.