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Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

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We developed low-cost, locally produced cell-free gene expression systems for rapid RNA detection. These systems match commercial performance, enabling accessible biosensing for disease surveillance.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Cell-free gene expression systems offer potential for field diagnostics.
  • Toehold switch-based RNA sensors enable detection of specific RNA molecules.
  • Commercial cell-free systems are costly and require cold chains, limiting accessibility in the Global South.

Purpose of the Study:

  • To develop and validate low-cost, locally producible cell-free gene expression systems for RNA sensing.
  • To enhance the stability of DNA templates in cell lysates for direct use with PCR products.
  • To create novel sensors for plant pathogens like Potato Virus Y (PVY).

Main Methods:

  • Utilized in-house cell lysates for cell-free gene expression.
  • Implemented CRISPR interference (CRISPRi) to knock down genes degrading linear DNA.
  • Designed and tested toehold switch-based RNA sensors for Potato Virus Y detection.

Main Results:

  • Achieved sensor performance comparable to commercial cell-free systems using in-house lysates.
  • Reduced sensor production costs by two orders of magnitude.
  • Demonstrated enhanced stability of linear DNA templates in optimized cell lysates.
  • Successfully developed novel sensors for Potato Virus Y.

Conclusions:

  • Locally produced cell-free systems offer a cost-effective alternative to commercial systems for RNA sensing.
  • CRISPRi optimization enhances the utility of cell lysates for rapid sensor design and screening.
  • This approach can decentralize biosensing capacity and improve global infectious disease surveillance.