Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation,ACS Chemical Biology

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Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2022-06-14 , DOI: 10.1021/acschembio.2c00101
Xiao-Han Liu ₁ , Bang-Rui Li ₁ , Zhan-Ming Ying ₁ , Li-Juan Tang ₁ , Fenglin Wang ₁ , Jian-Hui Jiang ₁

Affiliation

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.

中文翻译：

用于诱导型 CRISPR-dCas9 转录激活的小分子介导的分裂适体组装

诱导型CRISPR-dCas9转录系统已成为转录调控和传感的强大工具。在这里，我们开发了一种用于诱导型 CRISPR-dCas9 转录激活的小分子介导的分裂适体组装的新概念，从而可以对活细胞中的 S-腺苷甲硫氨酸 (SAM) 进行定量检测和成像。这种诱导型转录系统的设计方法是将分裂的 SAM 适体的一个片段整合到引导 RNA (gRNA) 并将另一个片段整合到 MS2 阵列中。 SAM 介导的分裂片段重新组装将 MCP 融合的转录激活剂招募到 gRNA-dCas9 复合物中，激活近红外荧光蛋白的表达以进行成像。我们证明这种诱导转录系统可以在活细胞中高灵敏度地定量检测 SAM。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，并且癌细胞中的 SAM 水平可以通过表观遗传抑制剂上调 MAT1A mRNA 来增加。这种分裂适体组装策略可以提供一种控制 CRISPR-dCas9 系统的新方法，从而能够根据活细胞中的内源代谢物进行条件转录调控。

更新日期：2022-06-14

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