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.

V 型和 VI 型 CRISPR–Cas 系统已被证明可以反式切割非特异性单链 DNA (ssDNA) 或单链 RNA (ssRNA)，但在使用单向导 RNA 的 II 型 CRISPR–Cas 系统中尚未观察到这种情况。我们在此表明​​，由 CRISPR RNA 和反式激活 CRISPR RNA 双 RNA 指导的 II 型 CRISPR-Cas9 系统对 ssDNA 和 ssRNA 底物均显示出 RuvC 结构域依赖性反式切割活性。 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。我们发现Cas9的反式切割活性可以被目标ssDNA、双链DNA和ssRNA激活。 Cas9与指导RNA和靶RNA复合物的晶体结构为靶RNA结合激活Cas9提供了结构基础。基于Cas9的反式切割活性和核酸扩增技术，我们开发了DNA激活Cas9检测和RNA激活Cas9检测的核酸检测平台，能够高灵敏度和特异性地检测DNA和RNA样本。