Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.
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Authors
Arce, Anibal
Guzman Chavez, Fernando
Gandini, Chiara
Puig, Juan
Matute, Tamara
Dalchau, Neil
Pardee, Keith
Federici, Fernán
Publication Date
2021Journal Title
Frontiers in Bioengineering and Biotechnology
ISSN
2296-4185
Publisher
Frontiers Media
Volume
9
Number
727584
Language
eng
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Arce, A., Guzman Chavez, F., Gandini, C., Puig, J., Matute, T., Haseloff, J., Dalchau, N., et al. (2021). Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.. Frontiers in Bioengineering and Biotechnology, 9 (727584) https://doi.org/10.3389/fbioe.2021.727584
Abstract
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.
Keywords
cell-free, decentralization, diagnostics, low-cost, toehold-sensor
Sponsorship
Engineering and Physical Sciences Research Council (EP/R014000/1)
Biotechnology and Biological Sciences Research Council (BB/L014130/1)
Identifiers
External DOI: https://doi.org/10.3389/fbioe.2021.727584
This record's URL: https://www.repository.cam.ac.uk/handle/1810/330010
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