Spherical Nucleic Acids as Modulators of CRISPR/Cas12a by a Steric Barrier Effect for Designing Versatile Biosensors

Discovery of CRISPR/Cas12a has revolutionized broad fields, including gene editing, molecular diagnosis, and biosensing. Flexible regulation of Cas12a activity is important for diverse CRISPR/Cas12a applications, especially for biosensing, but it still faces limitations and challenges. We find gold nanoparticles (AuNPs) modified with a single-stranded DNA activator create a huge steric barrier that strongly locks activators in a one-to-many manner and inhibits Cas12a activity. This finding offers a new way to modulate the activity of Cas12a for applications, such as designing versatile biosensors. We report a spherical nucleic acid (SNA)-modulating CRISPR/Cas12a (SNA-Cas) platform using SNAs as signal translators for target sensing. A stimuli-responsive SNA was constructed by modifying AuNPs with a DNA activator containing a specific trigger element, and the target triggers specific reactions (e.g., thiol-exchange chemical reaction and RNA-cleaving by DNAzymes) to release activators into solution. A free activator initiates trans-cleavage activity of CRISPR/Cas12a, scissoring fluorescent DNA reporters to produce amplified signals. To show proof of concept, we demonstrate SNA-Cas to sensitively detect diverse non-nucleic acid targets, including biological thiol cysteine, heavy metal Pb²⁺, and biomarkers of O⁶-methylguanine-DNA-methyltransferases (MGMT) and fat mass and obesity-related protein (FTO) demethylases. This work opens one door for SNA modulating CRISPR/Cas activity and shows great potential in designing versatile biosensors for detecting diverse targets.