CRISPRa screening with real world evidence identifies potassium channels as neuronal entry factors and druggable targets for SARS-CoV-2

  1. Chengkun Wang
  2. Ravi K. Dinesh
  3. Yuanhao Qu
  4. Arjun Rustagi
  5. Henry Cousins
  6. James Zengel
  7. Tianyi Zhang
  8. Nicholas Magazine
  9. Yinglong Guo
  10. Taryn Hall
  11. Aimee Beck
  12. Lucas Miecho Heilbroner
  13. Grace Peters-Schulze
  14. Aaron Wilk
  15. Luke Tso
  16. Elif Tokar Erdemic
  17. Kae Tanudtanud
  18. Sheng Ren
  19. Kathy Tzy-Hwa Tzeng
  20. Mengdi Wang
  21. Brooke Howitt
  22. Weishan Huang
  23. Jan Carette
  24. Russ Altman
  25. Catherine A. Blish
  26. Le Cong

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Abstract

Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been successful, there are no good treatments for those who are actively infected. While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from sensory organs and the brain are also susceptible to infection. While many patients suffer from diverse neurological symptoms, the virus’s neuronal entry remains mysterious. To discover host factors involved in SARS-CoV-2 viral entry, we performed CRISPR activation (CRISPRa) screens targeting all 6000+ human membrane proteins in cells with and without overexpression of ACE2 using Spike-pseudotyped lentiviruses. This unbiased gain-of-function screening identified both novel and previously validated host factors. Notably, newly found host factors have high expression in neuronal and immune cells, including potassium channel KCNA6, protease LGMN, and MHC-II component HLA-DPB1. We validated these factors using replication-competent SARS-CoV-2 infection assays. Notably, the overexpression of KCNA6 led to a marked increase in infection even in cells with undetectable levels of ACE2 expression. Analysis of human olfactory epithelium scRNA-seq data revealed that OLIG2+/TUJ1+ cells--previously identified as sites of infection in COVID-19 autopsy studies-- have high KCNA6 expression and minimal levels of ACE2. The presence of KCNA6 may thus explain sensory/neuronal aspects of COVID-19 symptoms. Further, we demonstrate that FDA-approved compound dalfampridine, an inhibitor of KCNA-family potassium channels, suppresses viral entry in a dosage-dependent manner. Finally, we identified common prescription drugs likely to modulate the top identified host factors, and performed a retrospective analysis of insurance claims of ~8 million patients. This large cohort study revealed a statistically significant association between top drug classes, particularly those targeting potassium channels, and COVID-19 severity. Taken together, the potassium channel KCNA6 facilitates neuronal entry of SARS-CoV-2 and is a promising target for drug repurposing and development.

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  1. Version published to 10.1101/2021.07.01.450475v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.07.01.450475: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    No key resources detected.

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A potential path to overcome these limitations is doing screens in multiple cell lines representing a variety of cell types, but this is laborious and necessitates prior knowledge of viral tropism. Our CRISPRa screening approach overcomes these limitations by selecting cell lines with limited or no susceptibility and allowing for the unbiased determination of factors that promote viral entry. We show here that previously unknown factors in a diverse set of tissues—such as neuronal (KCNA6), immune (HLA-DPB1, CD7), and cardiac (LGMN, EPHA4)—can stimulate SARS-CoV-2 viral entry. Most strikingly, we show that KCNA6, a voltage-gated potassium channel, potentiates SARS-CoV-2 entry in a cellular context where ACE2 expression is undetectable (Fig. 3E, 4H). Further, our additional tests demonstrated that, under such ACE2-null conditions, KCNA6 overexpression promoted pseudovirus infection when using different variants of the SARS-CoV-2 Spike, including a variant of concern B.1.351 (Beta variant, first identified in South Africa) (Fig. S12A). With minimal ACE2 expression, the Spike variants D614G and B.1.351 supported comparable ability as wild-type Spike to infect KCNA6-overexpressing cells in pseudoviral assay (Fig. S12B). This could be significant in light of reports that emerging SARS-CoV-2 variants are often less dependent on ACE2 binding, and resistant to antibody therapeutics 17,19,42. KCNA6 is highly expressed in OLIG2+ neuronal cells (Fig. 4F-H), which have been shown to be a ...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.07.01.450475v1 on bioRxiv