Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation.
Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation.
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DOI：
10.1021/acschembio.2c00101
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发表时间：
2022-06
影响因子：
4
通讯作者：
Xiaohui Liu;Bang-Rui Li;Zhan‐Ming Ying;Li-Juan Tang;Fenglin Wang;Jianhui Jiang
中科院分区：
文献类型：
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作者：
Xiaohui Liu;Bang-Rui Li;Zhan‐Ming Ying;Li-Juan Tang;Fenglin Wang;Jianhui Jiang
诱导型CRISPR-dCas9转录系统已成为转录调控和传感的有力工具。在这里，我们提出了一个小分子介导的分裂适体组装的新概念，用于诱导CRISPR-dCas9转录激活，允许在活细胞中定量检测和成像s -腺苷型甲硫氨酸（SAM）。这种诱导型转录系统是通过整合分裂的SAM适体的一个片段来引导RNA (gRNA)，另一个片段到MS2阵列来设计的。sam介导的分裂片段重组将mcp融合的转录激活子招募到gRNA-dCas9复合体上，激活近红外荧光蛋白的表达以进行成像。我们证明了这种诱导转录系统在活细胞中实现了高灵敏度的SAM定量检测。我们的系统表明，蛋氨酸腺苷转移酶1A （MAT1A）和MAT2A都可以催化活细胞中SAM的产生，并且表观遗传抑制剂可以通过上调MAT1A mRNA来增加癌细胞中的SAM水平。这种分裂适体组装策略可以提供一种新的方法来控制CRISPR-dCas9系统，从而在活细胞中响应内源性代谢物实现条件转录调控。
Inducible CRISPR-dCas9 transcription system has become a powerful tool for transcription regulation and sensing. Here, we develop a new concept of small-molecule-mediated split-aptamer assembly for inducible CRISPR-dCas9 transcription activation, allowing quantitative detection and imaging of S-adenosyl methionine (SAM) in live cells. This inducible transcription system is designed by integrating one fragment of a split SAM aptamer to guide RNA (gRNA) and the other to MS2 arrays. SAM-mediated reassembly of the split fragments recruits an MCP-fused transcription activator to the gRNA-dCas9 complex, activating the expression of a near-infrared fluorescent protein for imaging. We demonstrate that this inducible transcription system achieves quantitative detection of SAM with high sensitivity in live cells. Our system shows that methionine adenosyltransferase 1A (MAT1A) and MAT2A can both catalyze SAM production in live cells and the SAM levels in cancer cells can be increased via upregulation of MAT1A mRNA by epigenetic inhibitors. This split-aptamer assembly strategy could afford a new approach for controlling the CRISPR-dCas9 system, enabling conditional transcription regulation in response to endogenous metabolites in live cells.