RT Journal Article
SR Electronic
T1 Decentralizing cell-free RNA sensing with the use of low-cost cell extracts
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.05.29.446205
DO 10.1101/2021.05.29.446205
A1 Arce, Anibal
A1 Chavez, Fernando Guzman
A1 Gandini, Chiara
A1 Puig, Juan
A1 Matute, Tamara
A1 Haseloff, Jim
A1 Dalchau, Neil
A1 Molloy, Jenny
A1 Pardee, Keith
A1 Federici, Fernán
YR 2021
UL http://biorxiv.org/content/early/2021/05/30/2021.05.29.446205.abstract
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.