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

Abstract

The current global debacle of COVID-19, spelled by SARS-CoV-2 needs no elaboration. With incessant and constantly clambering number of deaths across various nations, the need of the hour is to develop readily deployable, fast, affordable detection assays and kits, yielding precise and consistent results as well as timely availability of efficacious anti-SARS-CoV-2 strategies to contain it. Conventionally employed real time PCR based technique for detection of the virus suffers from a couple of handicaps. Amongst other approaches, CRISPR based technology has ushered in new hopes. Recent efforts have been directed toward developing CRISPR/Cas based low-cost, rapid detection methods as well as development of one-pot assay platforms. The plausible application of CRISPR-Cas system to counteract the viral assault has also been assessed. The write up in this article mirrors the current status, the prospects and the practical snags of CRISPR/Cas technology for the detection and inactivation of the novel corona virus, SARS-CoV-2.

Keywords: COVID-19; CRISPR/Cas; RNA; SARS-CoV-2; diagnostics; viral inactivation.

FIGURE 1

CASdetec used for SARS-CoV-2 detection. (A) Fluorescence kinetics of sgRNA-3 for RdRp detection. E. coli cells bearing Blunt-SARS-CoV-RdRp or Blunt-SARS-CoV-2-RdRp were pre-incubated at 95 °C for 10 min and used as templates for RAA and CDetection. PAM sequences are colored in green, proto-spacers are colored in blue, base pair mismatches are colored in red. Error bars indicate standard errors of the mean (SEM), n = 3. RFU, relative fluorescence units. (B) Fluorescence kinetics of RdRp detection using 108 nM sgRNA-3. Plasmid bearing SARS-CoV-2-RdRp was serially diluted as shown in the legend. n = 2. ΔRn, ΔFluorescence, which refers to the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by ABI 7500. (C) Fluorescence kinetics of F1- and R1-based RdRp detection. SARS-CoV-2-RdRp RNA was serially diluted as shown in the legend. Error bars indicate (SEM), n = 3. (D) Evaluation of cross-reactivity. Plamids containing target RdRp region from six human epidemic coronaviruses were serially diluted as the shown in the legend. n = 2. (E) Detection of SARS-CoV-2 pseudovirus. Virus genome was extracted using the virus RNA extraction kit (spin column). SARS-CoV were diluted to 5 × 10⁵ copies/mL. n = 2. (F) Detection of SARS-CoV-2 pseudovirus. Virus was treated by direct lysis. SARS-CoV was diluted to 5 × 10⁵ copies/mL. n = 2. (G) CASdetec results could be directly observed under blue LED. 3 replicates of products from (C) were imaged upon blue LED illumination. (H) Schematics showing the workflow of CASdetec. Virus genome was extracted by kit or direct lysis. Target sequences were pre-amplified by isothermal amplification, followed was CDetection. Fluorescence signals were obtained either from fluorescence reader or direct observation under blue light (Reproduced from Guo et al., 2020, under the provisions of Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/, Copyright© The Author(s) 2020).

FIGURE 2

Highlights of PAC-MAN based strategy to target conserved sequences across coronaviruses and other pathogenic viruses (Reproduced from Abbott et al., 2020, based on the reuse-provisions of Elsevier’s COVID-19 Resource Centre, Copyright© 2020 Elsevier, Inc.).