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

Arce, Anibal 
Guzman Chavez, Fernando 
Gandini, Chiara 
Puig, Juan 
Matute, Tamara 

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Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

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cell-free, decentralization, diagnostics, low-cost, toehold-sensor
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Frontiers in Bioengineering and Biotechnology
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Frontiers Media
Engineering and Physical Sciences Research Council (EP/R014000/1)
Biotechnology and Biological Sciences Research Council (BB/L014130/1)