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 pointof-care solution to increase the distribution of COVID-19 surveillance.

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

“…Possible alternative explanations are variability in test performance or in representation of different portions of the viral RNA. Low detection efficiency could be a possible concern in other tests including those under development that are based on reverse transcription as a first step followed by DNA amplification and detection (Broughton et al, 2020;Rauch et al, 2020;Yan et al, 2020). Many RT-qPCR tests use RNA corresponding to about 1% of swab sample.…”

“…Further, the need for fluorescent oligonucleotide probes adds to the cost of the tests. A number of new diagnostic testing methods aimed at reducing the dependence on expensive equipment and kits that are in short supply have been proposed and are under development for deployment (Broughton et al, 2020;Rauch et al, 2020;Yan et al, 2020). However, given the enormous and widespread current need for diagnostic testing in diverse environments it is equally important to increase the utilization of existing research capabilities that have potential but are currently not being used for testing, through the use of tests that employ more widely available equipment and reagents using simple established methods.…”

Can CRISPR/Cas Technology Be a Felicitous Stratagem Against the COVID-19 Fiasco? Prospects and Hitches

“…However, assessing the sensitivity of the assay for clinical isolates and patient samples as well as demonstrating the specificity at species and subspecies levels of highly related viruses demand attention. In a similar vein, Rauch et al (2020) have recently proposed the application of CREST (Cas13-based, Rugged, Equitable, Scalable Testing) protocol as an orthogonal solution to augment testing potency for SARS-CoV-2. The approach involves the use of simple, easy to operate, portable thermocyclers and plastic-filter-based LED visualizers.…”

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2

“…Finally, since PEARL uses common reagents and it does not require expensive equipment or highly trained personnel, it can provide an accessible alternative for streamlining diagnostics in geographic areas that lack access to capital, specialized reagents, and professional laboratories. In view of these considerations, coupling PEARL to our recently developed CREST protocol for detection of SARS-CoV-2 genetic material 16 could allow streamlined, affordable, widespread testing, lowering the barrier of "luxury testing" in many regions of the world.…”