Jiyun Chen, Ying Chen, Linglong Huang, Xiaofeng Lin, Hong Chen, Wenwen Xiang & Liang Liu

Link: https://www.nature.com/articles/s41587-024-02255-7

Published: 29 May 2024

Abstract: The trans-cleavage activity of non-specific ssDNA or ssRNA is widely found in type V and type VI CRISPR-Cas systems, while not observed in type II CRISPR-Cas systems when guided with sgRNA. We show here that the type II CRISPR-Cas9 systems directed by crRNA and tracrRNA dual-RNAs show RuvC domain-dependent trans-cleavage activity for both ssDNA and ssRNA substrates.Cas9 possesses sequence preferences for trans-cleavage substrates, preferring to cleave T- or C-rich ssDNA substrates. We find that the trans-cleavage activity of Cas9 can be activated by target ssDNA, dsDNA, and ssRNA. We report the crystal structure of Cas9 in complex with guide RNA and target RNA, which provides the structural basis for the binding of target RNA to activate Cas9. Based on the trans-cleavage activity of Cas9 and nucleic acid amplification technology, we have developed DACD (DNA-activated Cas9 Detection) and RACD (RNA-activated Cas9 Detection) nucleic acid detection platforms. DACD and RACD are capable of detecting DNA and RNA samples with high sensitivity and specificity. Together, our study demonstrates the crRNA and tracrRNA-directed trans-cleavage activity of Cas9, highlighting the potential of Cas9 as a new, versatile molecular detection tool.