Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza

Original Article: Abbott TR, Dhamdhere G, Liu Y, et al. Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell. 2020;181(4):865-876.e12.

Author of summary: Alessio Silva; Reviewer: Marianna Coppola

PAC-MAN (prophylactic antiviral CRISPR in human cells), a Cas13-based strategy, can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus in human lung epithelial cells. The authors designed and screened CRISPR RNAs covering conserved viral regions and identified 6 crRNAs capable of targeting more than 90% of all coronaviruses. Therefore, PAC-MAN can be a potential pan-coronavirus inhibition strategy.

The authors propose a prophylactic class 2 type VI-D CRISPR-Cas13d customizable antiviral approach (PAC-MAN) capable of simultaneously degrading intracellular viral genomes and their resulting subgenomic RNA and mRNA templates in human cells, including SARS-CoV-2 and Influenza A virus (IAV), thus to provide broad virus protection.

Unfortunately, they were unable to use live SARS-CoV-2 strains, therefore performing their experiments with viral fragments and live H1N1 IAV as a proof of concept, infecting human A549 lung epithelial cells.

Using bioinformatics, they designed, screened and selected 40 crRNAs, with 20 each targeting highly conserved regions across many sequenced SARS-CoV-2 genomes, in particular the genes encoding for the RNA-dependent RNA polymerase (RdRP) and the nucleocapsid (N).

To validate Cas13d activity, they generated a stable A549 cell line expressing the enzyme. Therefore, they transfected or transduced one of the two reporters expressing synthesized fragments of SARS-CoV-2 fused to GFP (SARS-CoV-2-RdRP-F1 and SARS-CoV-2-RdRP-N-F2) together with pools of 4 crRNAs targeting either RdRP or N genes.

Ten groups (G1-G10) were tested and the results analysed 24h after via flow cytometry and qPCR, revealing that G4 targeting the F1 RdRP fragment was particularly effective and able to repress GFP. A strong decrease in GFP expression was also provoked by G5 and G6, targeting the F2 RdRP and N portions, respectively. Similar results were obtained with qPCR analysis. Besides, a high level of crRNA but not Cas13d concentration increased the cleaveage effectiveness.

Subsequently, they tested the Cas13d PAC-MAN on the H1N1 strain of IAV. Upon bioinformatic analysis, they identified 6 crRNAs targeting highly conserved regions for each of the 8 RNA segments of the IAV genome. Then, they transfected those pools in Cas13d A549 cells and infected them with an H1N1 IAV strain mNeon-tagged under stringent conditions. Optimal results were obtained with the pool targeting the segments 6 and 4, encoding for the neuraminidase and hemagglutinin proteins, respectively. Moreover, IAV replication was strongly inhibited at lower titers, suggesting a more effective application of Cas13d in preventing viral infection.

Afterwards, they analysed fully sequenced 91’600 IAV strains, finding out that 81 crRNAs can target them all, with a minimum group of 6 crRNAs covering 92% of IAV genomes, suggesting a potential pan-IAV inhibitory approach. They did the same analysis with all 3’051 coronavirus genomes known, revealing 6 crRNAs capable of targeting 91% of sequenced coronaviruses with no mismatches and 22 crRNAs able to target all of them. This expands the applications of CRISPR-Cas13 systems beyond diagnostics, with PAC-MAN potentially capable to target not only viruses that circulate in humans but also those found in animal reservoirs.

Future perspectives:

  • In vitro models infection with live SARS-CoV-2;
  • PAC-MAN inhibits viral sequences in vitro but in vivo efficacy, specificity, immunogenicity and delivery must be validated passing through preclinical models.


  • crRNAs must be validated for off-target effects;
  • PAC-MAN must be expressed in a certain percentage of cells to be effective;
  • Cas13d/crRNAs in vivo levels must be finely tuned;
  • Viral genome secondary structure and/or protein coatings may reduce inhibition efficiency.

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