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Membrane-Confined CRISPR-Cas12a Activation via Split Activator for Wash-Free Detection of Tumor-Specific
Yongan Ren1, Rui Wu1, Xu Yang1
1School of Chemical and Environment Science, Shaanxi University of Technology, Hanzhong 723001, China.
Abstract:
Current CRISPR-Cas12a biosensing systems for protein analysis rely on the release of a blocked activator from an aptamer through a competitive mechanism. However, the design of the complementary length between the activator and the aptamer involves a critical trade-off: excessively long sequences impede aptamer-target binding, whereas overly short sequences fail to activate Cas12a effectively. To overcome this limitation, we split the full-length Cas12a activator into two short single-stranded DNA fragments (S1 and S2; 10 nt each). S1 was specifically sequestered within the hairpin structure of an allosteric aptamer. The binding of the target protein to the aptamer triggered a conformational change, exposing S1 and thereby enabling its assembly with S2 to activate Cas12a. The strategy successfully preserved the binding affinity of the aptamer without compromising the efficiency of Cas12a activation. Furthermore, we anchored the Cas12a substrate to the membrane surface of extracellular vesicles (EVs) via hydrophobic interactions, localizing the cleavage reaction to the EVs interface and thereby significantly enhancing local catalytic efficiency. Finally, the strategy provided highly sensitive detection of tumor-specific EVs, not only accurately distinguishing cancer patients from healthy individuals (77.5% accuracy for early-stage and 100% for advanced-stage) but also holding potential for monitoring dynamic postoperative changes. Overall, our study provided a wash-free, one-pot detection platform for EVs analysis and established a new paradigm for CRISPR-based clinical diagnostics.
Insights
This study introduces a novel CRISPR-Cas12a biosensing platform for detecting tumor-specific extracellular vesicles (EVs). The innovative design enhances sensitivity and accuracy in cancer diagnostics.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- CRISPR Technology
Background:
- CRISPR-Cas12a biosensors for protein analysis face design limitations regarding activator-aptamer sequences.
- Optimizing sequence length is crucial for balancing aptamer-target binding and Cas12a activation efficiency.
Purpose of the Study:
- To develop an improved CRISPR-Cas12a system for sensitive extracellular vesicle (EV) detection.
- To overcome limitations in current CRISPR-based biosensing by redesigning the Cas12a activator mechanism.
- To enhance the localization and catalytic efficiency of the biosensing reaction on EVs.
Main Methods:
- Split the Cas12a activator into two single-stranded DNA fragments (S1 and S2) for improved aptamer binding and activation.
- Utilized an allosteric aptamer to sequester S1, releasing it upon target protein binding.
- Anchored the Cas12a substrate to extracellular vesicle membranes via hydrophobic interactions to enhance local catalytic efficiency.
Main Results:
- Achieved preserved aptamer binding affinity and efficient Cas12a activation with the split activator strategy.
- Demonstrated highly sensitive detection of tumor-specific EVs, distinguishing cancer patients from healthy individuals with high accuracy (77.5% early-stage, 100% advanced-stage).
- Showcased the potential for monitoring dynamic postoperative changes via EV analysis.
Conclusions:
- Developed a wash-free, one-pot detection platform for EV analysis using a novel CRISPR-Cas12a system.
- Established a new paradigm for CRISPR-based clinical diagnostics with enhanced sensitivity and specificity.
- The split-activator strategy and EV-anchoring approach offer a robust platform for future diagnostic applications.
