Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants

Abstract

The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.

Prevalence and Factors Associated with Parents’ Non-Intention to Vaccinate Their Children and Adolescents against COVID-19 in Latin America and the Caribbean

“…Regarding the differences in the non-intention to vaccinate among the countries, differences in the epidemiological situation, the wave or time of the pandemic in which they were found, and the proportion of people affected by COVID-19 in these countries must be taken into account. Likewise, the circulation of variants of the SARS-CoV-2 virus of the most significance, such as the alpha, beta, gamma, and delta variants [45], and especially the latter, could influence the decision to get vaccinated. As of 4 August 2021, this variant is present in 24 countries in the Americas region, and due to its greater transmissibility, mortality from this disease could increase, and health systems could collapse [46].…”

A Thermostable Cas12b fromBrevibacillusLeverages One-pot Detection of SARS-CoV-2 Variants of Concern

“…2f). We envision that this approach, with proper safety precautions, could be used for home-based testing, as demonstrated by others 3,4,13,20 . The portable multiplexing detection prototype was built with two wavelengths in the optical system: a LED working at 490/26 nm excitation for FITC, and a second LED working at 625/17 nm with the same length of impulse for Cy5 excitation.…”

“…Since the beginning of the COVID-19 pandemic, many strategies have been explored to develop rapid and sensitive detection kits to drive the diagnostics towards home-based testing [1][2][3][4][5] . Current gold-standard Reverse Transcription -quantitative Polymerase Chain Reaction (RT-qPCR) tests require sophisticated instrumentation as well as intensive labor training, and therefore become a major hurdle for deployment in remote areas.…”

CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout

“…Among several tools developed so far, CRISPR-based methods SHERLOCK, DETECTR, and FELUDA techniques have shown rapid, 100% accuracy, sensitivity, and high specificity detection of SARS-CoV-2 but are not quantitative as compared to the standard PCR method. Recently, researchers have slightly modified the above methods which can not only detect the different variants of the SARS-CoV-2 efficaciously but also can detect the viral load and generate absolute quantification within 1 h. (Kaminski et al 2021 ) These CRISPR-methods are named as follows: first, RAY (Rapid variant AssaY), an advanced version of FELUDA which uses multiplexing of crRNAs of a different mutated version of SARS-CoV-2 simultaneously accompanied with FnCAS9 (Kumar et al 2021 ); second, miSHERLOCK (Minimally Instrumented) which uses multiplexing of crRNAs of a mutated version of SARS-CoV-2 simultaneously accompanied with Cas12a, and addition to that, it requires diagnostic chamber which includes sample concentrator, incubator, and fluorescence reporter and also supported with a mobile application that automatically interprets and quantifies the mutated variants (de Puig et al 2021 ); third, RADICA (RApid DIgital Crispr Approach) which uses multiplexing of crRNAs of SARS-CoV-2, SARS-CoV, and MERS-CoV simultaneously accompanied with Cas12a followed by isothermal amplification of the extracted DNA and partitioning of the extracted DNA into a commercially available integrated chip. As the amplification begins, Cas12a cleaves the + ssDNA strand, and the fluorescence signal is detected by the detector and gives the absolute quantification of the virus particle (Wu et al 2021 ).…”