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.