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

ABSTRACT

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.