Open platform for the implementation of RNA sensing reactions in cell-free systems

Abstract

Synthetic biology seeks the development of programmable and predictable functions in biological systems. One of the latest advances in the field are the toehold switches: de-novo engineered regulators of gene expression that allow the translation of a gene only after the interaction with a cognate “trigger” RNA of a specific sequence.On the other hand, cell-free protein expression systems have been used for decades facilitating discoveries in molecular biology, and have played a critical role in the elucidation of the genetic code. More recently, cell-free systems have emerged as a tool for the engineering of genetic devices, bio-products, and biosensors, among many other applications. The expression of programmable toehold switch-based RNA sensors in cell-free systems has generated as promising platform for diagnostics. Cell-free toehold sensorscan be freeze-dried for room temperature transport to the point-of-need.These sensors, however, have been implemented using reconstituted PURE(Protein expression Using Reconstituted Elements) cell-free protein expres-sion systems that are difficult to source in Latin America, due to their pro-hibitively expensive commercial cost, and cold-chain shipping requirements(-80oC) from suppliers in the northern hemisphere.Here, we describe the implementation of RNA toehold sensors usingE. coli cell lysate-based cell-free protein expression systems, which can be pro-duced locally and reduce the cost of sensors by two orders of magnitude, while providing sensor performance comparable to commercial PURE cell-free systems. Further optimization of the cell extracts with a CRISPRistrategy enhanced the stability of linear dsDNAs, enabling the use of PCR products as a substrate for cell-free gene expression reactions including to hold sensors. As a proof-of-concept application, we used the tools de-veloped in this thesis to prototype and screen 8 novel RNA toehold sensors for the potato pathogen Potato Virus Y (PVY virus) that 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 diseases.