A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material

Abstract

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.

Paper-Based Biosensors: Frontiers in Point-of-Care Detection of COVID-19 Disease

“…However, PCR technique is more sensitive and can be used with widely available reagents, and the fluorescence readout is more reliable than the lateral flow readout. Therefore, attempts are in progress to use small, portable, battery-powered and inexpensive thermocyclers and fluorescence visualizers [111].…”

Recent progress on rapid SARS-CoV-2/COVID-19 detection by CRISPR-Cas13-based platforms

“…Rauch and colleagues [30] recently developed another CRISPR-based SARS-CoV-2 RNA detection method named CREST (Cas13-based, Rugged, Equitable, Scalable Testing). This method was developed as an option for recurrent testing that could overcome issues with the supply of reagents and specialized equipment, and that is easy to deploy at sites with minimal infrastructure [30] . CREST addresses three of the main hurdles that limit the scalability of Cas13-based testing at sites with a minimal supply of resources: 1) reagent accessibility, 2) equipment availability, and 3) cost.…”

“…CREST addresses three of the main hurdles that limit the scalability of Cas13-based testing at sites with a minimal supply of resources: 1) reagent accessibility, 2) equipment availability, and 3) cost. By taking advantage of widely available reagents (that are stable at room temperature), low-cost thermocyclers (instead of isothermal reactions), and easy-to-use fluorescent visualizers, this method bypasses the need for highly trained personnel and experienced molecular diagnostic laboratories [30] .…”

“…However, the amplification step uses cost-effective Taq polymerases and portable thermocyclers. This has been made possible by creating inexpensive, Bluetooth-enabled thermocyclers (which can even be powered by a 9 V battery and run using mobile device applications) and simple plastic-filter-based, LED visualizers [30] . In the CREST platform, transcription and Cas13 activity are followed by fluorescence detection of de-quenched poly-U cleavage reporter, visualized with a blue LED (~495 nm) and orange filter or other fluorescence detection system.…”

“…Using CREST, Rauch and colleagues [30] detected the presence of the N1, N2, and N3 sites in the SARS-CoV-2 nucleocapsid ( N ) gene, with an associated cost of reagents per test of US$ 5.90 (a single sample run in triplicate) compared to the PCR test cost of US$ 14 apiece. Thus, CREST is a relatively cheap format that allows portable diagnostic tests to assess suspected cases without sophisticated equipment.…”