Split Activator Assembly via Aptamer Binding Enables CRISPR‐Cas12a Activation With Non‐Nucleic Acid Targets

The programmability of CRISPR-Cas12a has enabled transformative advances in nucleic acid detection; however, extending its activation to non-nucleic-acid targets remains highly desirable. Here, we report a universal split-activator strategy that enables programmable Cas12a activation in response to a small molecule and a protein. In this approach, a canonical activator is divided into two fragments (p1 and p2), such that Cas12a activation occurs only upon their simultaneous assembly. We integrate this mechanism with aptamer-mediated conformation switching, wherein target binding destabilizes an aptamer-p2 duplex, releasing p2 to complete the activator complex and trigger Cas12a nuclease activity. Using this design, we demonstrate selective recognition of the small-molecule metabolite adenosine and the protein biomarker prostate-specific antigen (PSA), with high specificity against structurally related controls. Signal generation is achieved using fluorescent DNA nanoclusters (DFN-1), which undergo fluorescence decrease upon Cas12a-mediated degradation. The system exhibits concentration-dependent responses for both targets. Together, this work establishes a modular framework for coupling aptamer-based molecular recognition to programmable Cas12a activation, expanding the functional scope of CRISPR systems beyond nucleic acids and enabling new opportunities in CRISPR-Cas12a-mediated detection.