Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR/Cas9 screening and transcriptomics
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PROVIDER: PRJNA647903 | ENA |
REPOSITORIES: ENA
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Project description:The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness. As clinical trials are ongoing, the full side effect profile of remdesivir is not yet known. Prior clinical studies have identified adverse events, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR/Cas9 screening (data not provided here) and RNA sequencing, we identify specific genes and pathways implicated in remdesivir metabolism. We show that remdesivir treatment leads to a global repression of mitochondrial respiratory activity. Further, we show that loss of the mitochondrial nucleoside transporter SLC29A3 or the mitochondrial adenylate kinase AK2 provide 10-72-fold mitigation of remdesivir toxicity while conserving its antiviral potency. This work provides candidate gene targets to reduce remdesivir toxicity and provides a proof-of-principle for the use of CRISPR/Cas9 screening to elucidate mechanisms of drug cytotoxicity.
2020-09-08 | GSE154936 | GEO
Project description:The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness 1 . Prior clinical studies have identified adverse events 1,2 , and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments 7 , yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.
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Project description:CRISPR-Cas is an RNA-based defense system that enables prokaryotes to recognize invading foreign DNA by cognate crRNA guides and destroy it by CRISPR-associated Cas nucleases 1,2 . Elucidation of the interference mechanism of the Streptococcus pyogenes Type II CRISPR- Cas9 system has allowed for the successful repurposing of SpCas9 as a generic genome editing tool, with great promise for human gene therapy 3 . However, especially for therapeutic applications, some caution seems appropriate, because Cas9 systems from some human pathogens may induce a cytotoxic response via an unknown mechanism 4 . Here we show that when released in human cells, Cas9 nucleases from the pathogenic bacteria Campylobacter jejuni and S. pyogenes have the potential to cause severe DNA damage. In the absence of a CRISPR RNA guide, native Cas9 nucleases from both pathogens enter the host nucleus, where their presence leads to promiscuous double stranded DNA breaks (DSBs) and induction of cell death. DSB induction can be reduced to background levels either by saturation of CjCas9 and SpCas9 with crRNA guides or by inactivating their nuclease activity. Our results demonstrate that guide-free Cas9 of bacterial pathogens might play an important role in pathogenicity. Furthermore, we propose that saturating Cas9 with appropriate guide RNAs is crucial for efficient and safe therapeutic applications.
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