CRISPR-Cas system is a powerful technology, functioning as a nucleic acid-based adaptive immune system of bacteria and archaea against viruses or plasmids invasion. The discovery of this system has revolutionized the development of molecular biology, with broad applications in medicine and biotechnology.

Cas nucleases with trans-cleavage activity are more widely used in nucleic acid detection, represented by Cas12a. However, the specificity of Cas12a for single-base mismatch recognition is not high, which hinders its application in nucleic acid detection.

CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing with a better target recognition specificity, but its cleavage activity is lower than that of Cas12a.

what we did?

Based on the rational design strategy, we mutated the key amino acids on the helix α7 in CasΦ protein and introduced the hairpin-structured crRNA to construct a CRISPR-Cas system with high cleavage activity and good target recognition specificity.