Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector
Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector
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DOI：
10.1101/2021.09.13.460032
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发表时间：
2021-09
影响因子：
14.9
通讯作者：
S. Grüschow;C. Adamson;M. F. White
中科院分区：
文献类型：
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作者：
S. Grüschow;C. Adamson;M. F. White
III型CRISPR系统检测入侵的RNA，导致酶促Cas 10亚基的激活。Cas 10环化酶结构域产生环状寡腺苷酸（cOA）第二信使分子，激活多种效应核酸酶，降解核酸以提供免疫力。原噬菌体编码的Vibrio metoecus type III-B（VmeCmr）基因座未被表征，缺乏Cas 10中的HD核酸酶结构域，并编码NucC DNA核酸酶效应子，该效应子也被发现与基于环状寡核苷酸的抗噬菌体信号传导系统（CBASS）相关。在这里，我们证明了VmeCmr被靶RNA结合激活，产生环状三腺苷酸（cA 3），以刺激强大的NucC介导的DNA酶活性。VmeCmr的特异性被探测，揭示了RNA双链体的片段1和前间隔区侧翼序列（PFS）中特定核苷酸位置的重要性。我们利用这个可编程系统，以证明一个高度特异性和灵敏度的检测SARS-CoV-2病毒RNA的检测限（LoD）为2 fM的潜在使用商业板读取器没有任何外在的扩增步骤。灵敏度高度依赖于所使用的指导RNA，这表明靶RNA二级结构起着重要的作用，也可能与体内相关。
Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.