RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing
RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing
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DOI：
10.1021/acs.analchem.8b05238
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发表时间：
2019-03-19
影响因子：
7.4
通讯作者：
Li, Jinghong
中科院分区：
文献类型：
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作者：
Li, Yue;Teng, Xucong;Li, Jinghong
CRISPR/Cas9已经成为一种强大的基因组操作工具，对该系统的进一步工程设计使其能够根据外部信号进行精确调节，从而扩大其应用可能性，如生物传感或生物成像。然而，大多数刺激反应性CRISPR系统都是基于精心设计和工程设计的可诱导Cas9蛋白构建的，外部刺激仍然大多局限于小分子和光。为了构建更精确和易于构建的应答性CRISPR系统，并扩大其应答物种，我们寻求设计条件引导RNA，而不是Cas9蛋白，来介导与逻辑操作相对应的条件CRISPR。在这里，我们通过gRNA重构和支点介导的链位移构建mrna传感CRISPR，其中每个靶位可以独立控制。我们发现开关可以嵌入gRNA并用作RNA传感器，能够正交检测多个mRNA输入并提供CRISPR/Cas9响应输出。也构造了NOR和NAND逻辑门，证明了其正交性和可编程性。这一策略有望用于构建基因回路以检测内源性mrna并启动细胞反应。
CRISPR/Cas9 has already become a powerful tool for genomic manipulation, and further engineering of the system allows it to be precisely regulated in response to external signals, thus, broadening its application possibilities, such as biosensing or bioimaging. However, most stimuli responsive CRISPR systems are built based on elaborately designed and engineered inducible Cas9 proteins, and external stimuli are still mostly limited as small molecules and light. To construct more precise and easy-to-build responsive CRISPR systems and broaden their responsive species, we seek to engineer conditional guide RNA, rather than Cas9 protein, to mediate conditional CRISPR corresponding to logic operation. Here, we construct mRNA-sensing CRISPR by gRNA reconfiguration and toehold mediated strand displacement, in which each target site could be independently controlled. We show that switches can be embedded into the gRNA and used as RNA sensors, capable of detecting multiple mRNA inputs orthogonally and providing CRISPR/Cas9 response outputs. NOR and NAND logical gates are also constructed, demonstrating its orthogonality and programmability. This strategy promises potential uses in constructing genetic circuits to detect endogenous mRNAs and initiate cellular responses.