Genome-wide CRISPR screening used to identify essential factors of SARS-CoV-2 infection in humans

In a methodologically pioneering article published in the journal Genetics of nature, a team of Belgian scientists has discovered a set of coronavirus host factors that could be exploited to develop drugs against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but also potentially the zoonotic coronaviruses to come.

The 2019 coronavirus pandemic (COVID-19), caused by SARS-CoV-2, has resulted in a global health and economic crisis; however, while vaccines are now in use around the world, only a handful of effective drugs are currently available to treat patients with the disease.

Although remdesivir was initially seen as a promising candidate for severe cases of COVID-19, trials conducted by the World Health Organization (WHO) have shown that in reality it does not have a significant impact. on mortality. On the other hand, dexamethasone has been shown to reduce mortality by a third in critically ill people, mainly by suppressing the overactive immune response.

Yet dexamethasone only benefits severe cases with a relatively limited effect. Innovative, safe and effective treatment options are therefore urgently needed. In this regard, the development of broad-spectrum coronavirus drugs would not only help cope with the current pandemic, but could also be used to quickly contain potential zoonotic events in the future.

But to develop such drugs, a full understanding of the host factors that are used by coronaviruses to infect a cell is essential, as each step in the viral replication cycle can serve as an intervention target. Although the coronavirus entry stage has been described in detail, details of host-virus interaction in later parts of the viral life cycle are still elusive.

Therefore, a Belgian research group (led by Dr Jim Baggen of the Rega Institute in Louvain) used genome-wide CRISPR-based genetic screens (regularly spaced short palindromic repeats) to discern the factors exact host numbers needed for SARS. -CoV-2 and human coronavirus 229E (HCoV-229E) infection.

A genome-wide knockout screen based on CRISPR

In this study, the aforementioned CRISPR-based genome-wide knockout screening was performed in the human Huh7 cell line (derived from male hepatoma tissue) without the introduction of an exogenous receptor since the strain SARS-CoV -2 used in the study prompted the development of an obvious cytopathic effect in these cells.

In addition, tests have been carried out with both SARS-CoV-2 and much less pathogenic HCoV-229E which is endemic and causes the common cold. This, in turn, allowed the identification of not only specific host factors of SARS-CoV-2 and HCoV-229E, but also of HCoV-OC43 and coronaviruses.

To validate the results obtained, the researchers expressed a unique guide RNA targeting identified genes and known receptor genes in Huh7 cells. As a result, they were able to see whether the ablation of these genes affected the sensitivity of cells to the cytopathic effect induced by SARS-CoV-2 or HCoV-229E.

Common host factors for human coronaviruses

This study unveiled phosphoinositide 3-kinase (PI3K) type 3 as a common host factor for SARS-CoV-2, HCoV-229E, and HCoV-OC43, meaning that the small molecules that target this protein could be widely applicable as anti-coronavirus inhibitors.

In addition, the screens demonstrated that the lysosomal protein TMEM106B in fact serves as a key host factor that enables SARS-CoV-2 infection in primary lung cells and human cell lines of hepatic origin. In patients with COVID-19, unicellular RNA sequencing of airway cells has shown that TMEM106B expression correlates with SARS-CoV-2 infection.

The researchers also showed that TMEM41B – a new regulator of autophagy (intracellular degradation system) and lipid mobilization – is necessary for infection with HCoV-229E, and to a lesser extent for infection with SARS-CoV-2.

Inform future research efforts

In short, this methodologically robust research study revealed a plethora of coronavirus host factors that could be used in the development of drugs against SARS-CoV-2 infection, but also zoonotic coronavirus outbreaks that could occur in the future.

“The set of essential coronavirus host factors identified in our genome-wide screens will form a basis for studies unraveling the cellular pathways hijacked by these viruses,” say the study authors. “Additionally, these factors may serve as targets for medicinal chemistry efforts to counter the current COVID-19 pandemic or future outbreaks of pathogenic coronaviruses,” they add.

It is now clear that pathogenic coronaviruses tend to emerge periodically, which means they will continue to pose a threat to global public health beyond the current COVID-19 pandemic. As a result, carrying out basic research studies (similar to this one) aimed at developing broad anti-coronavirus inhibitors will be an essential step forward.