Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

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.

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts

“…We showed that ΔrecBCD extracts support rapid screening and characterization of genetic constructs for two relevant applications: toehold switch library characterization, and enzyme activity screening. While this manuscript was being prepared for submission, consistent with our findings a paper published by Arce et al showed that cell-free extract depleted of the RecBCD exonuclease by CRISPRi can be used for prototyping of toehold switch biosensors encoded on linear DNA templates 57 . Our method, by avoiding cloning and PCR purification, provided significant gains in time and cost.…”

Biotechnology Applications of Cell-Free Expression Systems

“…In the past, the high cost of cell-free reagents prohibited their use in biosensing. However, technological innovations in cell-free have lowered the cost of preparing extracts that can be optimized for sensing applications [23,55]. Cell-free biosensors can be optimized to detect a range of possible analytes, and multiple biosensing strategies are compatible with a cell-free platform [23].…”

Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

“…Cost calculations and limitations. Comparative analysis of the E. coli cell-free protein expression system-based colorimetric RNA diagnostics with other approaches, with respect to commercial PURExpress, homemade OnePot PURE system 39,40 and E. coli extracts 33,41 . All costs are provided in EUR (euro) currency (with 1.1xUSD=EUR estimate), per total reaction volume.…”

“…We calculated the lysate preparation costs based on previous publications that were rigorously detailed 33,41 . In comparison to ref.…”