Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas12a is known for cleaving single-stranded DNA (ssDNA).
• ssDNA-templated probes have diagnostic potential but are limited in scope.
• Cas12a's activity on double-stranded DNA (dsDNA) substrates is not well-understood.

Purpose of the Study:

• To investigate CRISPR-Cas12a's trans-cleavage activity on dsDNA substrates.
• To develop and evaluate novel dsDNA-templated fluorescent probes for Cas12a.
• To expand the application range of Cas12a-based diagnostics.

Main Methods:

• Design and synthesis of dsDNA substrates with varying characteristics.
• Utilizing fluorescence spectroscopy to monitor Cas12a trans-cleavage activity.
• Optimizing experimental conditions including salt concentration and target DNA levels.

Main Results:

• A dsDNA substrate (probe-full) demonstrated effective Cas12a trans-cleavage.
• Performance of the dsDNA probe was superior to nicked dsDNA variants.
• Cas12a activity was successfully programmed for specific viral targets (TCSV, HepBV).
• High sensitivity (10 pM) and specificity were achieved in target detection.

Conclusions:

• CRISPR-Cas12a's trans-cleavage is not restricted to ssDNA substrates.
• Cas12a-based diagnostics can be effectively extended to utilize dsDNA substrates.
• This expands the versatility and application of CRISPR-Cas12a technology.