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High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells

Author

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  • Min Zhang

    (Tsinghua University)

  • Xue Zhang

    (Tsinghua University)

  • Yongyue Xu

    (Tsinghua University)

  • Yanhui Xiang

    (Chinese Academy of Sciences)

  • Bo Zhang

    (Tsinghua University)

  • Zhen Xie

    (Tsinghua University)

  • Qiong Wu

    (Tsinghua University)

  • Chunbo Lou

    (Chinese Academy of Sciences)

Abstract

RNAs and their encoded proteins intricately regulate diverse cell types and states within the human body. Dysregulated RNA expressions or mutations can lead to various diseased cell states, including tumorigenesis. Detecting and manipulating these endogenous RNAs offers significant promise for restoring healthy cell states and targeting tumors both in research and clinical contexts. This study presents an RNA-IN and RNA-OUT genetic circuit capable dynamically sensing and manipulating any RNA target in a programmable manner. The RNA-IN module employes a programmable CRISPR-associated protease (CASP) complex for RNA detection, while the RNA-OUT module utilizes an engineered protease-responsive dCas9-VPR activator. Additionally, the CASP module can detect point mutations by harnessing an uncovered dual-nucleotide synergistic switching effect within the CASP complex, resulting in the amplification of point-mutation signals from initially undetectable levels (1.5-fold) to a remarkable 94-fold. We successfully showcase the circuit’s ability to rewire endogenous RNA-IN signals to activate endogenous progesterone biosynthesis pathway, dynamically monitor adipogenic differentiation of mesenchymal stem cells (MSCs) and the epithelial-to-mesenchmal trans-differentiation, as well as selective killing of tumor cells. The programmable RNA-IN and RNA-OUT circuit exhibits tremendous potential for applications in gene therapy, biosensing and design of synthetic regulatory networks.

Suggested Citation

  • Min Zhang & Xue Zhang & Yongyue Xu & Yanhui Xiang & Bo Zhang & Zhen Xie & Qiong Wu & Chunbo Lou, 2024. "High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-52962-7
    DOI: 10.1038/s41467-024-52962-7
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