crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.

Area of Science:

• Molecular Biology
• Biotechnology
• Nucleic Acid Chemistry

Background:

• CRISPR-Cas12a offers powerful molecular diagnostics with signal amplification.
• Its continuous activity limits temporal control in complex assays.
• Precise nuclease regulation is crucial for advanced diagnostic applications.

Purpose of the Study:

• To develop a reversible and programmable method for controlling CRISPR-Cas12a activity.
• To engineer the crRNA scaffold as a conformational switch for Cas12a regulation.
• To enhance temporal programmability and nuclease control in Cas12a-based detection systems.

Main Methods:

• Repurposing crRNA scaffold secondary structure as a conformational switch.
• Utilizing tunable-length DNA blockers to induce and reverse scaffold structural changes.
• Implementing strand displacement mechanisms for on-demand Cas12a activity restoration.
• Assessing single nucleotide variant (SNV) discrimination and compatibility with isothermal amplification.

Main Results:

• Demonstrated length-dependent disruption and remodeling of crRNA scaffold secondary structure using DNA blockers.
• Achieved reversible inactivation and on-demand reactivation of LbCas12a activity.
• Showcased improved SNV discrimination and one-pot assay compatibility with high analytical sensitivity.
• Successfully detected Klebsiella pneumoniae and Mycobacterium tuberculosis using the developed regulatory framework.

Conclusions:

• The crRNA scaffold can be rewired as a universal and programmable switch to modulate CRISPR-Cas12a activity.
• This regulatory framework enables time-resolved and on-demand nuclease control for advanced molecular diagnostics.
• The approach offers mechanistic insights for engineering CRISPR-based assays with enhanced precision and versatility.