School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, China.
Biotechnol Bioeng. 2021 Aug;118(8):3037-3045. doi: 10.1002/bit.27813. Epub 2021 May 24.
RNA-guided CRISPR (RNA-targeting clustered regularly interspaced short palindromic repeats) effector Cas13d is the smallest Class II subtype VI proteins identified so far. Here, two recently identified Cas13d effectors from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) were characterized and applied for sensitive nucleic acid detection. We demonstrated that the special target triggered collateral cleavage of these two Cas13d orthologs could provide rapid target RNA detection in picomolar range and then the tolerance for mismatch between crRNA and target RNA was characterized as well. Finally, an additional single mismatch was introduced into crRNA to enhance the two Cas13d orthologs mediated detection of low variant allele fraction, 0.1% T790M. Overall, this study demonstrated that both EsCas13d and RspCas13d could robustly detect target RNA carrying special single-nucleotide variation with high specificity and sensitivity, thereby providing newly qualified machinery in toolbox for efficient molecular diagnostics.
RNA 导向的 CRISPR（RNA 靶向的成簇规律间隔短回文重复序列）效应 Cas13d 是迄今为止发现的最小的 II 类 VI 型蛋白。在这里，我们对两种最近从互养菌（Eubacterium siraeum，Es）和瘤胃球菌属（Ruminococcus sp.，Rsp）中鉴定出的 Cas13d 效应蛋白进行了表征，并将其应用于灵敏的核酸检测。我们证明，特殊的靶标触发这两种 Cas13d 同源物的旁侧切割，可以在皮摩尔范围内快速检测靶标 RNA，然后还对 crRNA 和靶标 RNA 之间的错配容忍度进行了表征。最后，在 crRNA 中引入一个额外的单碱基错配，以增强两种 Cas13d 同源物对低变异等位基因分数（0.1% T790M）的检测。总的来说，这项研究表明，EsCas13d 和 RspCas13d 都可以高度特异性和灵敏度地稳健地检测携带特殊单核苷酸变异的靶标 RNA，从而为高效分子诊断提供了新的合格工具。
