Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology , Tsinghua University , Beijing 100084 , China.
School of Pharmaceutical Sciences , Zhengzhou University , Zhengzhou 450001 , China.
Anal Chem. 2019 Mar 19;91(6):3989-3996. doi: 10.1021/acs.analchem.8b05238. Epub 2019 Mar 11.
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.
CRISPR/Cas9 已经成为基因组操作的强大工具，进一步的系统工程使其能够对外界信号进行精确调控，从而拓宽其应用可能性，如生物传感或生物成像。然而，大多数响应性 CRISPR 系统都是基于精心设计和工程化的诱导型 Cas9 蛋白构建的，外部刺激仍然主要局限于小分子和光。为了构建更精确和易于构建的响应性 CRISPR 系统，并拓宽其响应物种，我们试图工程化条件性向导 RNA，而不是 Cas9 蛋白，以介导与逻辑运算相对应的条件性 CRISPR。在这里，我们通过 gRNA 重构和触发介导的链置换构建了 mRNA 感应型 CRISPR，其中每个靶位点都可以独立控制。我们表明，开关可以嵌入 gRNA 中并用作 RNA 传感器，能够正交检测多个 mRNA 输入，并提供 CRISPR/Cas9 响应输出。还构建了 NOR 和 NAND 逻辑门，证明了其正交性和可编程性。该策略有望用于构建遗传电路，以检测内源性 mRNA 并引发细胞反应。
