CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.

Area of Science:

• CRISPR-based diagnostics
• Nucleic acid sensing technologies
• Molecular diagnostics

Background:

• CRISPR technologies offer rapid, accurate nucleic acid detection but face challenges like PAM sequence requirements and limited multiplexing.
• Existing CRISPR diagnostics struggle with labile RNA inputs and require specific targeting sequences.

Purpose of the Study:

• To develop a versatile CRISPR-based nucleic acid barcoding technology.
• To create a diagnostic platform overcoming current CRISPR limitations for broad applicability.
• To demonstrate the diagnostic's efficacy in detecting infectious diseases.

Main Methods:

• Developed Functionalized Amplification CRISPR Tracing (FACT) compatible with Cas12a and Cas13a for sequence-agnostic detection.
• Integrated FACT with the Reprogrammable PAIRing (RePAIR) system to link CRISPR activation to protein expression.
• Created the FACTOR diagnostic platform by combining FACT and RePAIR.
• Applied FACTOR to detect viral infections in honey bees.

Main Results:

• FACTOR enables diagnostic outputs based on cis- and trans-cleavage from any nucleic acid sequence.
• The Reprogrammable PAIRing system successfully links CRISPR-Cas12a activation to protein expression.
• FACTOR demonstrated high specificity and accuracy in detecting honey bee viral infections.
• The study showcases the potential for sensing any nucleic acid of interest.

Conclusions:

• FACTOR represents a significant advancement in CRISPR-based diagnostics, overcoming key implementation challenges.
• The FACTOR platform offers a versatile and accurate solution for detecting diverse nucleic acid targets.
• This technology holds promise for broad applications in diagnostics, including infectious disease surveillance.