Anti-CRISPR Protein Regulates CRISPR/Cas12a Fusogenic-Nanovesicle-Based Platform for Extracellular...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nanomedicine

Background:

• Detecting non-nucleic acid targets in extracellular vesicles (EVs) is challenging due to isolation difficulties and low molecule abundance.
• Existing methods often suffer from signal degradation and insufficient output intensity, hindering accurate analysis.

Purpose of the Study:

• To develop a high-efficiency in-vesicle analysis strategy for detecting EV-encapsulated protein biomarkers.
• To overcome limitations in EV isolation and low target molecule abundance for improved signal transduction.

Main Methods:

• Aptamer-mediated membrane fusion and a "locked-activated" CRISPR-Cas12a-AcrVA1 (LACA) system were integrated.
• A yly12-aptamer-functionalized nanovesicle encapsulating the LACA system (yly12-lipo@Cas12a) was fabricated as an in-vesicle bioanalytical platform.

Main Results:

• The platform demonstrated high specificity and sensitivity in detecting EV-carrying protein biomarkers within 2.5 hours.
• Clinical analysis showed excellent differentiation between patients and healthy controls with an AUC of 0.965.
• The aptamer specificity and AcrVA1's regulatory function enhanced Cas12a activity for precise detection.

Conclusions:

• The proposed yly12-lipo@Cas12a nanovesicle platform offers a versatile and efficient solution for in-vesicle bioanalysis.
• This technology holds significant potential for the sensitive detection of EV protein biomarkers and disease diagnosis in clinical settings.