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. 2024 Sep 28;16(10):1539.
doi: 10.3390/v16101539.

A CRISPR-Cas13b System Degrades SARS-CoV and SARS-CoV-2 RNA In Vitro

Klara Andersson  1   2 , Ani Azatyan  2 , Martin Ekenberg  2 , Gözde Güçlüler  2 , Laura Sardon Puig  2 , Marjo Puumalainen  2 , Theodor Pramer  2 , Vanessa M Monteil  1   3 , Ali Mirazimi  1   3   4
Affiliations

A CRISPR-Cas13b System Degrades SARS-CoV and SARS-CoV-2 RNA In Vitro

Klara Andersson et al. Viruses. .

Abstract

In a time of climate change, population growth, and globalization, the risk of viral spread has significantly increased. The 21st century has already witnessed outbreaks of Severe Acute Respiratory Syndrome virus (SARS-CoV), Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2), Ebola virus and Influenza virus, among others. Viruses rapidly adapt and evade human immune systems, complicating the development of effective antiviral countermeasures. Consequently, the need for novel antivirals resilient to viral mutations is urgent. In this study, we developed a CRISPR-Cas13b system to target SARS-CoV-2. Interestingly, this system was also efficient against SARS-CoV, demonstrating broad-spectrum potential. Our findings highlight CRISPR-Cas13b as a promising tool for antiviral therapeutics, underscoring its potential in RNA-virus-associated pandemic responses.

Keywords: CRISPR-Cas13b; SARS-CoV; SARS-CoV-2; antiviral development.

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Conflict of interest statement

T.P. is the CEO of Biomedrex Genetics. M.P., A.A., M.E., G.G., and L.S.P. are employed by Biomedrex Genetics.

Figures

Figure 1
Figure 1
Overview of the selection and filtering of the most suited gRNAs to silence SARS-CoV-2. The gRNAs are filtered based on their target site conservation and relevance, secondary structure, host genome overlap, and direct repeat integrity. A total of 20 guide RNAs were chosen to be tested in vitro.
Figure 2
Figure 2
Single guide RNAs (gRNAs) silencing live SARS-CoV-2. (a) Scheme of the workflow for the screening of top-ranked gRNAs, including transfections of the plasmids encoding gRNA and Cas13b, infections with SARS-CoV-2, and analysis with qPCR. (b) The single gRNA silencing of SARS-CoV-2. n = 3, where n is the independent biological experiments. Statistical analysis performed with Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
gRNAs tested in pairs; 50 ng of each gRNA plasmid tested together compared to 50 ng of the gRNAs tested individually. (a) NSP12-targeting gRNA together with NSP5 gRNA, (b) NSP12 gRNA together with S gRNA, and (c) NSP12 gRNA together with N gRNA. n = 3, where n is the independent biological experiments. Statistical analysis performed with Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns. non-significant.
Figure 4
Figure 4
CRISPR-Cas13b designed to target SARS-CoV-2 efficacy on SARS-CoV. Statistical analysis performed with Student’s t-test. n = 3, where n is the independent biological experiments. * p < 0.05, **** p < 0.0001, ns: non-significant.
See this image and copyright information in PMC

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