Fusing Allosteric Ribozymes with CRISPR‐Cas12a for Efficient Diagnostics of Small Molecule Targets

Abstract The CRISPR‐Cas systems are adopted as powerful molecular tools for not only genetic manipulation but also point‐of‐care diagnostics. However, methods to enable diagnostics of non‐nucleic‐acid targets with these systems are still limited. Herein, by fusing ligand‐dependent allosteric ribozymes with CRISPR‐Cas12a, a derived CRISPR‐Cas system is created for efficient quantitative analysis of non‐nucleic‐acid targets in 1–2 h. On two different small molecules, the system's generality, reliability and accuracy is demonstrated, and show that the well operability of this system can enable high‐throughput detection of a small molecule in blood samples. The system can be further converted to rely on allosteric deoxyribozyme instead of allosteric ribozyme to recognize non‐nucleic‐acid targets and transduce the signal to CRISPR‐Cas12a for amplification, likely making it easier for storage and more consistent in data generation as DNA possess a stability advantage over RNA. This (deoxy)ribozyme‐assisted CRISPR‐Cas12a system anticipates that it can facilitate bioanalysis in various scientific and clinical settings and further drive the development of clinical translation.