PT  - JOURNAL ARTICLE
AU  - Arce, Anibal
AU  - Chavez, Fernando Guzman
AU  - Gandini, Chiara
AU  - Puig, Juan
AU  - Matute, Tamara
AU  - Haseloff, Jim
AU  - Dalchau, Neil
AU  - Molloy, Jenny
AU  - Pardee, Keith
AU  - Federici, Fernán
TI  - Decentralizing cell-free RNA sensing with the use of low-cost cell extracts
AID  - 10.1101/2021.05.29.446205
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.29.446205
4099  - http://biorxiv.org/content/early/2021/05/30/2021.05.29.446205.short
4100  - http://biorxiv.org/content/early/2021/05/30/2021.05.29.446205.full
AB  - 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 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. Here, we describe the implementation of RNA toehold switch-based sensors using E. coli cell lysate-based cell-free protein expression systems, which can be produced locally and reduce the cost of sensors by two orders of magnitude. We then demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize use of these lysates with a CRISPRi strategy to enhance the stability of linear DNAs, enabling 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.Competing Interest StatementThe authors have declared no competing interest.