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用于精确、可调控制基因表达的微小RNA电路模块。

miRNA circuit modules for precise, tunable control of gene expression.

作者信息

Du Rongrong, Flynn Michael J, Honsa Monique, Jungmann Ralf, Elowitz Michael B

机构信息

Howard Hughes Medical Institute and Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Max Planck Institute of Biochemistry, Martinsried, Germany; Faculty of Physics, Ludwig Maximilian University, Munich, Germany.

出版信息

bioRxiv. 2024 Mar 12:2024.03.12.583048. doi: 10.1101/2024.03.12.583048.

Abstract

The ability to express transgenes at specified levels is critical for understanding cellular behaviors, and for applications in gene and cell therapy. Transfection, viral vectors, and other gene delivery methods produce varying protein expression levels, with limited quantitative control, while targeted knock-in and stable selection are inefficient and slow. Active compensation mechanisms can improve precision, but the need for additional proteins or lack of tunability have prevented their widespread use. Here, we introduce a toolkit of compact, synthetic miRNA-based circuit modules that provide precise, tunable control of transgenes across diverse cell types. These circuits, termed DIMMERs (Dosage-Invariant miRNA-Mediated Expression Regulators) use multivalent miRNA regulatory interactions within an incoherent feed-forward loop architecture to achieve nearly uniform protein expression over more than two orders of magnitude variation in underlying gene dosages or transcription rates. They also allow coarse and fine control of expression, and are portable, functioning across diverse cell types. In addition, a heuristic miRNA design algorithm enables the creation of orthogonal circuit variants that independently control multiple genes in the same cell. These circuits allowed dramatically improved CRISPR imaging, and super-resolution imaging of EGFR receptors with transient transfections. The toolbox provided here should allow precise, tunable, dosage-invariant expression for research, gene therapy, and other biotechnology applications.

摘要

在特定水平上表达转基因的能力对于理解细胞行为以及基因和细胞治疗应用至关重要。转染、病毒载体和其他基因递送方法会产生不同的蛋白质表达水平,定量控制有限,而靶向敲入和稳定筛选效率低下且速度缓慢。主动补偿机制可以提高精度,但对额外蛋白质的需求或缺乏可调性阻碍了它们的广泛应用。在这里,我们介绍了一套基于紧凑的合成miRNA的电路模块工具包,可在多种细胞类型中对转基因进行精确、可调的控制。这些电路被称为DIMMERs(剂量不变的miRNA介导的表达调节器),它们在非相干前馈环结构中利用多价miRNA调节相互作用,在基础基因剂量或转录速率超过两个数量级的变化范围内实现几乎均匀的蛋白质表达。它们还允许对表达进行粗略和精细控制,并且具有可移植性,能在多种细胞类型中发挥作用。此外,一种启发式miRNA设计算法能够创建正交电路变体,在同一细胞中独立控制多个基因。这些电路极大地改善了CRISPR成像以及通过瞬时转染对EGFR受体进行超分辨率成像。这里提供的工具包应能为研究、基因治疗和其他生物技术应用实现精确、可调、剂量不变的表达。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f87d/10979901/23b6c3d8123b/nihpp-2024.03.12.583048v1-f0001.jpg

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