厦门大学Liang Liu团队近期取得重要工作进展，他们研究提出，DNA或RNA靶标结合能激活Cas9的反式核酸酶活性。相关研究成果2024年5月29日在线发表于《自然—生物技术》杂志上。

据介绍，V型和VI型CRISPR–Cas系统已被证明能反式切割非特异性单链DNA（ssDNA）或单链RNA（ssRNA），但在使用单引导RNA的II型CRISPR-Cas系统中尚未观察到这一点。

研究人员表明，由CRISPR RNA和反式激活的CRISPR-RNA双RNA引导的II型CRISPR–Cas9系统，对ssDNA和ssRNA底物都显示出RuvC结构域依赖性的反式切割活性。Cas9对反式切割底物具有序列偏好，更倾向于切割富含T或C的ssDNA底物。研究人员发现，Cas9的反式切割活性可以被靶ssDNA、双链DNA和ssRNA激活。Cas9与引导RNA和靶RNA复合的晶体结构为靶RNA结合激活Cas9提供了结构基础。

总之，基于Cas9的反式切割活性和核酸扩增技术，研究人员开发了DNA激活Cas9检测和RNA激活Cas9检测的核酸检测平台，能够对DNA和RNA样品进行高灵敏度和特异性的检测。

附：英文原文

Title: Trans-nuclease activity of Cas9 activated by DNA or RNA target binding

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

Issue&Volume: 2024-05-29

Abstract: Type V and type VI CRISPR–Cas systems have been shown to cleave nonspecific single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) in trans, but this has not been observed in type II CRISPR–Cas systems using single guide RNA. We show here that the type II CRISPR–Cas9 systems directed by CRISPR RNA and trans-activating CRISPR RNA 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, double-stranded DNA and ssRNA. The crystal structure of Cas9 in complex with guide RNA and target RNA provides a 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 develop the nucleic acid detection platforms DNA-activated Cas9 detection and RNA-activated Cas9 detection, which are capable of detecting DNA and RNA samples with high sensitivity and specificity.

DOI: 10.1038/s41587-024-02255-7

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