New CRISPR-Cas13 Approach Inhibits SARS-CoV-2 Virus

A new CRISPR-based antiviral strategy targeting the SARS-CoV-2 virus appears to effectively degrade RNA. The CRISPR-Cas13 approach called PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) also proved effective against H1N1 human influenza A, another coronavirus, in human lung epithelial cells. With these findings, the researchers from Stanford University who developed the technique raise the possibility of developing a pan-coronavirus intervention against respiratory viruses. Their research is available as a pre-publication report in Cell.

The team set out with the goal of identifying a system that might be effective against a wide variety of viral illnesses. They first determined that only six CRISPR RNAs are able to target 91 percent of 3,051 sequenced coronaviruses. PAC-MAN was developed by targeting and screening conserved and functional RNAs targeting the SARS-CoV-2 virus.

“Altogether, the PAC-MAN-T6 can target all known human coronaviruses with broad coverage against other animal coronaviruses,” the authors write.

One important notable caveat to this research is that it was not conducted on live SARS-CoV-2 strains. Instead, the researchers relied on synthesized fragments of the virus, as well as with live infection using an H1N1 influenza A virus strain in human lung epithelial cells

Targeting the positive-sense RNA

The researchers first assumed that the SARS-CoV-2 life cycle is likely similar to the closely related coronavirus that causes SARS. These viruses replicate by entering the cell, releasing its RNA genome into the cytoplasm, and synthesizing negative-sense RNAs from which viral mRNAs and a new copy of the positive sense viral genome are synthesized.

This CRISPR approach recognizes and degrades the intracellular viral genome and its resulting viral mRNAs. The theory is that targeting the positive-sense genome and viral mRNAs to simultaneously degrade viral genome templates for replication and viral gene expression would inhibit viral replication.

Cas13 Variant Chosen for Pan-Virus Inhibition in Human Cells

The team chose the class 2 type VI-D CRISPR-Cas13d system derived from Ruminococcus flavefaciens XPD3002, a recently discovered RNA-guided RNA endonuclease. Cas13d employs CRISPR-associated RNAs that contain a customizable 22-nucleotide spacer sequence that can direct the Cas13d protein to specific RNA molecules for targeted RNA degradation.

Cas13d is known to possess high catalytic activity in human cells. Because of its small size (967 amino acids), high specificity, and strong catalytic activity, the researchers chose Cas13d rather than other Cas13 proteins to target and destroy RNA viruses including SARS-CoV-2 and Influenza A virus.

The team targeted highly conserved regions of the SARS-CoV-2 viral genome to target with Cas13d. These regions encode the RdRP and Nucleocapsid proteins, known to be essential for coronavirus replication and function. The authors suggest that inhibiting the proteins could have an outsized effect on disabling virus production and function, in addition to reducing viral load through degradation of the viral genome itself.

When targeting influenza A virus, the CRISPR RNAs that showed the best viral inhibition target the conserved ends of the segment S6 that encodes NA, a viral surface protein essential for mediating budding of new virions.

The research team also drew on the recent work with SARS-CoV-2 genomes showing that there are different subtypes (L and S) of the virus with different viral signature sequences. To see if the CRISPR RNAs can target all L and S strains, they tested it against  a set of 1,087 recently sequenced SARS-CoV-2 sequences from Global Initiative on Sharing All Influenza Data (GISAID).  They learned that it targets 1,083 out of 1,087 (99.6%) SARS-CoV-2 genomes suggesting that it can effectively target different SARS-CoV-2 strains.

An Enticing Proof-of-Concept

“Through the use of crRNA pools targeting different regions of the same virus or different strains of coronavirus, this system could possibly buffer against viral evolution and escape as well as be used to protect against future related pathogenic viruses,” the authors write. “While there are remaining hurdles to overcome before this strategy can be used clinically, PAC-MAN has the potential to become a new antiviral strategy.”

The authors stress that PAC-MAN is currently a proof-of-concept antiviral strategy for robustly and broadly targeting conserved viral sequences with Cas13, and a few important steps are required before it can be tested in clinical trials to possibly treat COVID-19.

At the top of the list is the need to test and validate the technique with live SARS-CoV-2 viruses, both in human cells and in preclinical animal models. The CRISPR RNAs that are selected to be tested therapeutically will also need to be evaluated for off-target effects. And, perhaps the most significant challenge is that any possible therapeutic uses will require an effective and safe in vivo delivery method into human respiratory tract cells.

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