Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.
Anibal Arce,Anibal Arce,Fernando Guzman Chavez,Chiara Gandini,Juan Puig,Juan Puig,Tamara Matute,Tamara Matute,Jim Haseloff,Neil Dalchau,Jenny Molloy,Keith Pardee,Fernán Federici +12 more
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TLDR
In this article, the authors 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, and demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems.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.read more
Citations
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Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples
Margot Karlikow,Severino Jefferson Ribeiro da Silva,Yuxiu Guo,Seray Cicek,Larissa Krokovsky,P J Homme,Yi-hong Xiong,Talia Xu,María Angélica Calderón-Peláez,Sigrid Camacho-Ortega,Duo Ma,Jurandy Júnior Ferraz de Magalhães,Bárbara Nayane Rosário Fernandes Souza,Diego Guerra de Albuquerque Cabral,Katariina Jaenes,Polina Sutyrina,Thomas C. Ferrante,Andrea D Benítez,Victoria Nipaz,Patricio Ponce,Darius G. Rackus,James J Collins,Marcelo Paiva,Jaime E. Castellanos,Varsovia Cevallos,Alexander A. Green,Constância Flávia Junqueira Ayres,Lindomar Pena,Keith M. Pardee +28 more
TL;DR: The results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America were reported in this paper .
Journal ArticleDOI
Constructing Cell-Free Expression Systems for Low-Cost Access
Fernando Guzmán-Chávez,Aníbal Arce,Abhinav Adhikari,Sandra Vadhin,José Antonio Pedroza-Garcia,Chiara Gandini,James W. Ajioka,Jenny Molloy,Sobeida Sánchez-Nieto,Jeffrey D. Varner,Fernán Federici,Jim Haseloff +11 more
TL;DR: This work deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts, which can produce desiccated extracts that are 203–424-fold cheaper than commercial versions.
Journal ArticleDOI
Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts
TL;DR: In this paper , the ΔrecBCD exonuclease gene cluster was deleted from the Escherichia coli BL21 genome and the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies.
Journal ArticleDOI
A Low-Cost, Thermostable, Cell-Free Protein Synthesis Platform for On-Demand Production of Conjugate Vaccines
TL;DR: In this paper , the authors identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability and a low-cost energy substrate.
Journal ArticleDOI
Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application
Shuying Liu,David Duplat,Paula Benitez-Bolivar,Cielo León,Stephany Villota,Eliana Veloz-Villavicencio,Valentina Arévalo,Katariina Jaenes,Yuxiu Guo,Seray Cicek,Lucas Robinson,Philippos Peidis,Joel Pearson,J. Woodgett,Tony Mazzulli,Patricio Ponce,Silvia Restrepo,John Mario González,Adriana Bernal,Marcela Guevara-Suarez,Keith M. Pardee,Varsovia Cevallos,Camila González,Rod Bremner +23 more
TL;DR: The utility of replacing thermocyclers with a portable PoC device (FluoroPLUM) and the combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.
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