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Authors: Timothy R. Abbott, et al. Link to paper: https://www.cell.com/pb-assets/products/coronavirus/CELL_CELL-D-20-00736.pdf

Journal/ Pre-Print: Cell (pre-proof of accepted peer-reviewed article)

Key Words: CRISPR, Cas13, SARS-COV-2, Influenza A, Anti-viral strategy

RESEARCH HIGHLIGHTS

1. Describe PAC-MAN, a CRISPR-Cas13d based antiviral approach for degrading viral RNA in infected cells that can be adapted to multiple viruses

2. By targeting highly conserved regions of SARS-CoV2 and Influenza A virus RNA, they successfully degrade SARS-CoV2 RNA and reduce Influenza A virus protein expression in vitro.

3. A computational approach identifies highly-conserved regions across all coronaviruses that could be targeted to develop a pan-coronavirus treatment.

SUMMARY 

This study describes a CRISPR-Cas13d based strategy to inhibit viral replication by degrading viral RNA in infected cells and applies it to two model viruses: SARS-CoV2 and Influenza A (IAV). Using computational analysis of available SARS-CoV2 sequences, they developed CRISPR-RNAs (crRNA) targeting highly-conserved regions in the RdRp and N genes, allowing to degrade both genomic and sub-genomic viral RNAs. They assessed their efficacy against SARS-CoV2 RNA fragments also expressing GFP transfected or transduced into Cas13d stably expressing cells. To use a live virus, they developed crRNA targeting IAV and assessed their efficiency in decreasing gene expression of a reporter mNeonGreen IAV during infection. Further bioinformatics analysis found 6 crRNAs that could target 91% of all human and animal coronaviruses. While this paper doesn’t offer a direct solution to the current COVID-19 pandemic, it highlights a potential pan-coronavirus therapy for the future.

IMPACT FOR SARS-COV2/COVID19 RESEARCH EFFORTS

Treat SARS-CoV2/COVID19 positive individuals

· This study highlights a CRISPR-Cas13d strategy as a potential treatment for individuals infected with SARS-CoV-2 and potentially other future coronavirus infections. But it is very far from the clinic as this approach poses a lot of off-target and delivery issues.

STUDY TYPE

· In silico study / bioinformatics study

· In vitro study

STRENGTHS AND LIMITATIONS OF THE PAPER

Novelty: Describes a new CRISPR-Cas13d based antiviral approach that can be adapted to multiple viruses including SARS-CoV2.

Standing in the field: This paper is part of a body of literature that uses CRISPR-based systems to degrade viral RNAs within infected cells but is the first study applying it to SARS-CoV2.

Appropriate statistics: No, ‘two-sided t test with unequal variance’ used throughout, despite most data lacking normality. These reviewers did not have sufficient knowledge of computational approaches to make comments on these parts of the manuscript.

Viral model used: SARS-CoV-2 RNA fragments also expressing sfGFP, no replicating virus

H1N1 A/Puerto Rico/8/1934 strain with mNeon fluorescent gene in segment 4.

Translatability: This study is a proof-of-concept, which is far from clinical application. As discussed by the authors, many issues remain, e.g. delivering the Cas13d and crRNAs into respiratory tract cells in humans, and potential problems with immunogenicity of Cas13d (the authors highlight that this has already been an issue with Cas9). Potentially in the future this could be a useful technique, however it is currently at a very early stage and will not be ready to use during the current pandemic.

Main limitations:

· This study should show efficacy against a live SARS-CoV-2. The secondary structures of the viral RNA and its availability for Cas13d degradation will most likely be very different between the reporter system used here and live virus infection. Influenza A virus is too different from SARS-CoV2 and is not sufficient to extrapolate conclusions about live SARS-CoV-2.

· The authors used a human respiratory epithelial cell line stably expressing Cas13d (A549). Despite near 100% expression of Cas13d in the target cells, the strategy does not lead to 100% RNA degradation or inhibition of protein expression. Since Cas13d expression wouldn’t be 100% in humans, this may not be very effective.

· Here, the cell line expresses Cas13d and is transfected with the crRNA before viral infection. This suggests that this strategy can only be used as a prophylactic.

· Some experiments lack important control conditions, e.g. SARS-CoV-2 fragments without the target sequence showing that the crRNA is not targeting any other part of the construct (e.g. GFP).

· The authors use computational approaches to identify 6 crRNAs that can target 91% of all coronaviruses but do not show any in vitro experiments to confirm that these RNAs: (1) do inhibit various coronavirus such as common cold CoVs, (2) do not show off-target effects, (3) do not present issues with secondary structures. Out of 5 pools of crRNA tested on each SARS-CoV2 gene, only one per gene showed an inhibition >70% in their reporter assay. This pool of 6 crRNA might therefore not work.