Cas12a-assisted split crRNA complex for analysis and detection of diverse entities

Area of Science:

• Biotechnology
• Molecular Biology
• Diagnostics

Background:

• The Cas12a-crRNA system is a potent tool for nucleic acid diagnostics due to its sequence-specific DNA binding and collateral ssDNA cleavage activity.
• Current limitations restrict Cas12a applications to nucleic acid targets, hindering its development into a universal detection platform.

Purpose of the Study:

• To systematically investigate parameters for activating a Cas12a-split crRNA system and establish design guidelines.
• To develop CASCADE, an adaptable platform extending Cas12a detection capabilities beyond nucleic acids.
• To demonstrate CASCADE's utility for diverse targets and its integration with lateral flow assays for point-of-care applications.

Main Methods:

• Systematic exploration of Cas12a-split crRNA activation parameters.
• Development and validation of the CASCADE platform using microRNA as a model.
• Testing CASCADE for non-nucleic acid targets including small molecules and proteins.
• Integration of CASCADE with lateral flow assay (LFA) for portable detection.

Main Results:

• Established comprehensive design guidelines for Cas12a-split crRNA systems.
• Validated CASCADE's sensitivity, specificity, and mismatch discrimination for microRNA detection.
• Successfully detected non-nucleic acid targets (tobramycin, kanamycin, biotin, tetracycline repressor protein) with high sensitivity and specificity.
• Demonstrated user-friendly, on-site detection capabilities through LFA integration.

Conclusions:

• CASCADE is an adaptable platform that significantly broadens the application scope of the Cas12a system beyond nucleic acids.
• The developed system shows promise for sensitive and specific detection of diverse targets.
• Integration with LFA enables portable, user-friendly diagnostics for point-of-care or environmental monitoring.