key: cord-0955549-hh37bsfp
authors: Dobrindt, Kristina; Hoagland, Daisy A.; Seah, Carina; Kassim, Bibi; O’Shea, Callan P.; Murphy, Aleta; Iskhakova, Marina; Fernando, Michael B.; Powell, Samuel K.; Deans, P. J. Michael; Javidfar, Ben; Peter, Cyril; Møller, Rasmus; Uhl, Skyler A.; Garcia, Meilin Fernandez; Kimura, Masaki; Iwasawa, Kentaro; Crary, John F.; Kotton, Darrell N.; Takebe, Takanori; Huckins, Laura M.; tenOever, Benjamin R.; Akbarian, Schahram; Brennand, Kristen J.
title: Common genetic variation in humans impacts in vitro susceptibility to SARS-CoV-2 infection
date: 2021-02-13
journal: Stem Cell Reports
DOI: 10.1016/j.stemcr.2021.02.010
sha: f0d961717beb282d036d53790962eeefa8b9735c
doc_id: 955549
cord_uid: hh37bsfp

The host response to SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, demonstrates significant inter-individual variability. In addition to showing more disease in males, the elderly, and individuals with underlying comorbidities, SARS-CoV-2 can seemingly render healthy individuals with profound clinical complications. We hypothesize that, in addition to viral load and host antibody repertoire, host genetic variants impact vulnerability to infection. Here we apply human induced pluripotent stem cell (hiPSC)-based models and CRISPR-engineering to explore the host genetics of SARS-CoV-2. We demonstrate that a single nucleotide polymorphism (rs4702), common in the population, and located in the 3’UTR of the protease FURIN, impacts alveolar and neuron infection by SARS-CoV-2 in vitro. Thus, we provide a proof-of-principle finding that common genetic variation can impact viral infection, and thus contribute to clinical heterogeneity in COVID-19. Ongoing genetic studies will help to identify high-risk individuals, predict clinical complications, and facilitate the discovery of drugs.

A growing number of human genetic variants have been identified that contribute to enhanced susceptibility or resistance to viral diseases (Kenney et al., 2017) . Genetic discoveries related to virus-host interactions implicate genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins in disease outcome. Such insights have been made across a range of pathogenic viruses, including influenza A virus (Ciancanelli et al., 2015) , norovirus (Lindesmith et al., 2003) , and human immunodeficiency virus (Samson et al., 1996) .

There is marked variability between individuals in response to SARS-CoV-2 infection, with outcomes ranging from asymptomatic to critical (6.1%) (Guan et al., 2020) . There is an urgent need to explore the molecular mechanisms underlying this unexpected clinical heterogeneity. A range of factors in addition to viral load can impact outcomes, from genetic predisposition (e.g. blood type (Ellinghaus et al., 2020) , mutations in immune signaling receptors (van der Made et al., 2020) , or inborn errors of type I IFN immunity ) to immune repertoire (e.g. cross-reacting antibodies from other coronaviruses (Yuan et al., 2020) or autoantibodies to interferon ).

Although the host response to SARS-CoV-2 has been defined in lung cultures and tissue (Karczewski et al., 2020) , it remains unclear the full extent to which this varies across cell types and donors. Given the complicated host immune response to SARS-CoV-2 (Blanco-Melo et al., 2020) , the expression of a number of human genes likely modulates infection.

In an effort to determine whether host variants that may influence viral entry might contribute to the heterogeneity of COVID-19 symptoms, we assessed human variants of FURIN. The spike glycoprotein that resides on the surface of the SARS-CoV-2 virion facilitates viral entry into target cells by engaging host angiotensin-converting enzyme 2 (ACE2) as the entry receptor, and host cellular serine protease TMPRSS2 for spike protein priming Yan et al., 2020) . More controversial is the extent to which host BSG, a transmembrane glycoprotein, serves as a secondary entry receptor (Shilts et al., 2021; Wang et al., 2020) . The SARS-CoV-2 spike protein incorporates a four amino acid insertion that introduces a proposed cleavage site for FURIN, a host membrane-bound proprotease convertase, potentially resulting in priming of spike protein before viral exit from the cell (Coutard et al., 2020; Wrapp et al., 2020) . In contrast to SARS-CoV-2, the SARS-CoV spike protein lacks this FURIN-cleavage site, thus requiring cleavage to facilitate subsequent cell entry (Walls et al., 2020) . This theoretical hijacking of host FURIN activity is one possible explanation for the increased infectivity of SARS-CoV-2. Not only do these host genes show tissue-specific and cell-type-specific expression patterns, but each is associated with non-coding common genetic variants thought to regulate expression of each gene (GTEx Consortium et al., 2017) . Here we test the hypothesis that variability in the expression of host genes between cell types and individuals predicts susceptibility to infection. Given that CRISPR (clusters of regularly interspaced short palindromic repeats)-based allelic conversion of rs4702 in human induced pluripotent stem cells (hiPSCs) from AA to GG decreased FURIN mRNA levels, reduced neurite outgrowth, and altered neuronal activity in induced glutamatergic neurons (Schrode et al., 2019) , here we consider the impact of FURIN expression and genotype on SARS-CoV-2 infection across hiPSC-derived lung, intestinal and brain models. Our findings suggest that uncovering the genetic underpinnings of SARS-CoV-2 outcomes may help predict susceptibility for COVID-19, as well as facilitate precision treatment and prevention approaches.

A subset of COVID-19 patients present with neurological symptoms (Mao et al., 2020) . COVID-19 has been associated with acute disseminated encephalomyelitis (ADEM) (Pilotto et al., 2020) , midand long-term sequelae including classical Guillain-Barré syndrome (Toscano et al., 2020) , and COVID-19-associated delirium (McLoughlin et al., 2020) . After hospital discharge, an alarmingly high fraction of patients, as high as 33%, suffer from a dysexecutive syndrome consisting of inattention, disorientation, or poorly organized movements in response to command (Helms et al., 2020) . It is critical to resolve whether potential SARS-CoV-2 pathological mechanisms include direct infection of brain cells, consistent with reports of neurotropism and trans-synaptic spread by other coronaviruses , or simply reflect endothelial injury, vascular coagulopathy, and/or diffuse neuroinflammatory processes.

A variety of evidence reveals that human brain tissue and cultured neurons express critical host genes required for viral entry. RNA transcripts and protein for ACE2, FURIN, and BSG were detected in post-mortem adult human brain (prefrontal cortex (PFC) and midbrain substantia nigra (SN)) ( Fig.  1A,B; Supplemental Fig. 1) . Likewise, RNA-sequencing data from hiPSC-derived neural progenitor cells (NPCs) (Hoffman et al., 2017) , NGN2-induced glutamatergic neurons (Zhang et al., 2013) , ASCL1/DLX2-induced GABAergic neurons (Yang et al., 2017) , ASCL1/NURR1/LMX1A-induced dopaminergic neurons (Dobrindt et al., 2020) , as well as differentiated forebrain neurons (FB, comprised of a mixture of neurons and astrocytes), confirmed expression of many candidate host genes (Fig. 1A) . Although induced GABAergic and dopaminergic neurons expression higher levels of some host genes, NGN2-induced glutamatergic neurons were used for all neuronal infections because the eQTL effect associated with common variant rs4702 is best characterized in NGN2induced glutamatergic neurons neurons (Schrode et al., 2019) . Expression of FURIN, ACE2, BSG, and TMPRSS2 in NGN2-induced glutamatergic neurons, in mock (uninfected) and SARS-CoV-2 infected populations, was confirmed by qPCR, normalized to lung alveolosphere samples to facilitate inter-cell type comparisons of expression, with FURIN and BSG being most prominent and ACE2 being very low and almost undetectable (Fig. 1C) .

To determine neuronal susceptibility to SARS-CoV-2 virus in vitro, human NGN2-induced glutamatergic neurons derived from two independent donors were infected with SARS-CoV-2 virus at low multiplicity of infection (MOI 0.05). Glutamatergic neurons were susceptible to SARS-CoV-2 virus infection, rapidly increasing expression of subgenomic viral RNA (vRNA) nucleocapsid transcript by qPCR (148,124-fold, p-value <0.0001) within 24 hours, as well as increased Interferon β (IFNB1), a type-I interferon canonical of the innate immune response to viral infection (Fig. 1D) . Additionally, protein immunohistochemistry for SARS-CoV-2 nucleocapsid (N) was detected within 48 hours of infection (MOI of 0.05) (Fig. 1E) . It is important to note that the utilized qPCR primers assay subgenomic transcripts sequences only and do not detect the initial inoculum (see methods for explanation). At higher titers (MOI 1, 24 hours), immunoblot against N protein was consistent with SARS-CoV-2 replication in neurons (Fig. 1F) .

To further explore the susceptibility of the in vivo brain to SARS-CoV-2, we analyzed RNAsequencing datasets of brains of 3-5 week old male Golden Syrian hamsters infected intranasally with SARS-CoV-2 or a phosphate buffered saline (PBS) control (GSE161200) (Hoagland et al., 2021) . By RNA sequencing, we identified high levels of SARS-CoV-2 host gene expression in hamster brain tissue, with notably high levels of FURIN and BSG/CD147 ( Fig. 2A) . SARS-CoV-2 infection resulted in widespread up-and downregulation of transcripts (Fig. 2B) , notably upregulation of interferonassociated genes within 24 hours, an effect that subsided somewhat by day 8 (Fig. 2C) . Gene set enrichment analysis of differentially expressed genes was performed across a collection of 698 neural-themed gene sets subdivided into 8 categories (Schrode et al., 2019) . Notably, significantly upregulated gene sets (false discovery rate (FDR) < 5%) included those related to abnormal neuronal morphology and development, membrane trafficking and abnormal synaptic transmission (clustered hierarchically by significance in (Fig. 2D) , while downregulated gene sets included ion channel and neurotransmitter signaling pathways (Fig. 2E) . Nonetheless, alignment of the transcriptomic data to the SARS-CoV-2 genome failed to detect reads for virus beyond background, suggesting that inflammatory changes in gene expression persist with SARS-CoV-2 exposure despite lack of robust viral replication in vivo.

Altogether, these analyses suggest that although neuronal infection by SARS-CoV-2 can occur in response to high MOI in vitro, it may occur less frequently under clinically relevant conditions in vivo.

SARS-CoV-2 infects hiPSC-derived lung alveolospheres Han et al., 2021; and intestinal organoids (Lamers et al., 2020; Yang et al., 2020; Zhou et al., 2020) , particularly after dissociation; here we similarly apply published step-wise differentiation protocols for distal lung alveolar type 2-like epithelium (hereafter "alveolar cells") (Jacob et al., 2019) and intestinal organoids (Koike et al., 2019; Zhang et al., 2018) .

Alveolospheres formed after 30 days (Fig. 3A) robustly express the endothelial marker EPCAM as well as prosurfactant protein C (SFTPC) and NKX2-1 (Fig. 3B, D) . Alveolar cells were also detected to express host genes associated with SARS-CoV-2 infection (Fig. 3C) , including ACE2 (Fig. 3B) .

Consistent with other studies, we observed infection by SARS-CoV-2 of EPCAM and NKX2-1-positive lung alveolospheres derived from two donors across three infections on protein level (MOI 0.5, 24 hours) (Fig. 3D ) and on RNA level (MOI 0.1 and 0.5, 24 hours) (Fig. 3E) . Dissociated alveolar cells showed detectable SARS-CoV-2 N protein (MOI of 0.1, 24 hours) (Fig. 3F) . A plaque assay confirms effective viral production from the alveolospheres 24hpi at an MOI of 0.1 (Supplemental Fig. 2 ).

Intestinal organoids form within 24 days and express CDX2 and KRT20 protein, as well as ACE2 (Fig. 4A ). Similar to alveolospheres, EPCAM-positive intestinal organoids (one donor) also showed SARS-CoV-2 infection on protein level (MOI 0.5 and 1, 48 hours) ( Fig. 4B ) and on RNA level (MOI 0.05 and 0.5, 24 hours) (Fig. 4D) . Intestinal organoids express high levels of FURIN, ACE2 and BSG, but lower levels of TMPRSS2, relative to alveolospheres (Fig. 4C) . Dissociated intestinal cells showed detectable SARS-CoV-2 N protein (MOI of 0.05, 48 hours) (Fig. 4E) .

We optimized MOIs (neuron: 0.05, alveolosphere: 0.1, intestine: 0.05) and exposure times (neuron: 24 hours for RNA, 48 hours for protein; alveolosphere: 24 hours; intestine: 24 hours for RNA, 48 hours for protein) for subsequent studies of host gene and variant effects, towards maximizing cell viability and detection of SARS-CoV-2 nucleocapsid (N) protein.

To test the functional impact of decreasing expression of critical host genes, we applied a shRNA strategy to knock down expression of ACE2, FURIN, BSG and TMPRSS2 in neurons (Fig. 5A , B: FURIN (expression reduced to 56.9%, p-value <0.00045), ACE2 (expression reduced to 20%, p-value 0.0001), BSG (expression reduced to 37.8%, p-value <0.0001) and TMPRSS2 (expression reduced to 32.9%, p-value 0.002374)), lung alveolar cells ( Fig. 5D : FURIN (expression reduced to 14.5%, pvalue 0.0208), ACE2 (expression reduced to 21.1%, p-value 0.002), BSG (expression reduced to 16.5%, p-value <0.0001) and TMPRSS2 (expression reduced to 13.8%, p-value <0.0001)) and intestinal cells ( Fig. 5G : FURIN (expression reduced to 27.6%, p-value 0.0063), ACE2 (expression reduced to 28.4%, p-value 0.0323)and BSG (expression reduced to 13.4%, p-value 0.0434)) TMPRSS2 knock-down in intestinal cells was inconsistent due to extremely low host transcript expression and could not be included. Observed knockdown did not significantly differ across cell types or donors. Moreover, susceptibility to infection was quantified by qPCR for subgenomic vRNA nucleocapsid transcript and immunocytochemistry with high-content imaging for N protein. reduced to 49.7%, p-value <0.0001), ACE2 (expression reduced to 33.3%, p-value <0.0001and BSG (expression reduced to 11.1%, p-value <0.0001)). These findings were confirmed through quantification of fluorescence via high-content imaging in dissociated alveolar cells for N protein, which revealed decreased total infected cells compared to scrambled control (MOI of 0.1, 24 hours): shFURIN (N protein reduced to 50.7%, p-value <0.0001), shACE2 (N protein reduced to 10.7%, pvalue <0.0001), shBSG (N protein reduced to 20.1%, p-value <0.0001) and TMPRSS2 (N protein reduced to 63.2%, p-value <0.0001) (Fig. 5F) . Consistent with this, knockdown of FURIN, ACE2 and BSG in dissociated organoid intestinal cells led to a decreased number of N-positive SARS-CoV-2 infected cells through quantification of fluorescence via high-content imaging (MOI 0.5 after 48 hours): shFURIN (N protein reduced to 0.42%, p-value <0.0001), shACE2 (N protein reduced to 10.05%, p-value <0.0001) and shBSG (N protein reduced to 12.87%, p-value <0.0001) (Fig. 5I) . It is possible that the reduced percentage of N-positive cell in the scrambled controls with higher SARS-CoV-2 infection (MOI = 0.5) resulted from increased in cell death in alveolar cells, perhaps due to increased cellular stress from the combination of SARS-CoV-2 infection, shRNA transduction and antibiotic selection. The SARS-CoV-2 alone control (not shown), did indeed exhibit a higher infection rate at MOI 0.5. Since the infection rate of neurons on ICC level was extremely low, high-content imaging analysis of neurons could not be included.

Critically, shRNA-mediated knockdown of host genes resulted in large changes in their endogenous expression, which may not well model subtle changes in gene expression resulting from common variation between individual people but can serve here as positive controls to negatively affect SARS-CoV-2 entry and replication.

To achieve more physiologically relevant changes in gene expression, CRISPR-based allelic conversion was used to generate isogenic hiPSCs that differed at a single non-coding regulatory single nucleotide polymorphism (SNP) at the FURIN locus (rs4702, NC_000015.10:g.90883330G>A). This SNP was predicted to be a quantitative trait loci (eQTL) in the human brain (Fromer et al., 2016) and NGN2-induced glutamatergic neurons (Forrest et al., 2017) , and fine-mapping analysis identified a single putative causal cis-eQTL (probability = 0.94) (Schrode et al., 2019) . rs4702 was empirically validated to regulate FURIN expression, neuronal outgrowth and neuronal activity following allelic conversion from AA to GG in NGN2-induced glutamatergic neurons (Schrode et al., 2019) . Here we evaluate the regulatory effect of this common SNP, determining the extent to which rs4702 regulates FURIN expression and SARS-CoV-2 infection in lung cells and neurons (Fig. 6) .

rs4702 GG alveolospheres exhibit a reduced FURIN expression (-0.327-fold, p-value 0.0347) (Fig.  6B ) and demonstrated reduced genotype-dependent SARS-CoV-2 infection across both donors (-0.95-fold, p-value 0.0215) (Fig. 6A,C) . In 7-day-old CRISPR-edited rs4702 GG NGN2-induced glutamatergic neurons, we observed decreased FURIN expression relative to AA neurons, from two independent donors (-0.74-fold, p-value <0.0001) (Fig 6E) , consistent with (Schrode et al., 2019) . Both rs4702 AA and rs4702 GG neurons were infected by SARS-CoV-2 (MOI 0.05) at 48 hours ( Fig.  6D) , trending towards decreased SARS-CoV-2 infection in GG neurons by qPCR for subgenomic vRNA nucleocapsid transcript after 24 hours (-0.72-fold, p-value 0.0416) (Fig. 6F) . Altogether, these results indicate that reduced FURIN levels mediated by rs4702 result in decreased SARS-CoV-2 infection.

Overall, these findings suggest not only that gross manipulations of host genes can impact SARS-CoV-2 infection in lung and brain cells, but also that the more subtle gene expression changes associated with genetic common variants are sufficient to impact viral infection levels.

We apply a hiPSC-based model to explore the genetics of cell-type-specific host response to SARS-CoV-2. First, we report infection of post-mitotic human neurons by SARS-CoV-2. Second, we further demonstrate functional validation of the impact of host genes (ACE2, FURIN, BSG, and TMPRSS2) and genetic variation (rs4702) on SARS-CoV-2 infection in human neurons, lung and intestinal cells.

Our results highlight the importance of FURIN as a mediator for SARS-CoV-2 infection and further demonstrate that a common variant, rs4702, located in the 3'-UTR of the FURIN gene, and with allele frequency estimates from G=0.34 to 0.45 (Tryka et al., 2014) , is capable of influencing SARS-CoV-2 infection in vitro. More specifically, CRISPR/Cas9-mediated allelic conversion (from AA to GG) at the common variant rs4702 resulted in decreased neuronal and alveolar expression of the cis-gene target FURIN, and reduced SARS-CoV-2 infection. Our isogenic hiPSC-based strategy provides a proof-ofprinciple demonstration that common human genetic variation may directly impact SARS-CoV-2 infection.

The ability of SARS-CoV-2 to infect neurons observed here is consistent with other studies, which include reports of infection of human neurons and astrocytes using monolayer and organoid approaches (Jacob et al., 2020; Ramani et al., 2020; Song et al., 2021) , highlighting greater and productive infection in choroid plexus epithelial cells (Jacob et al., 2020) and less infection in NPCs (Ramani et al., 2020; Song et al., 2021) . One study comparing different hiPSC-derived cell types was able to detect infection in dopaminergic, but not cortical neurons, using an MOI of 0.01 on protein level (Yang et al., 2020) , thus a lower titer and a less sensitive detection method than used in our study, which are potentially less clinically relevant. Although it remains possible that neurons and/or glia in the brain do become infected in vivo, we see no evidence that infected brain cells consistently produce mature virions in our hamster model. The inflammatory signature we observe might be triggered by viral products and debris that make their way into the circulatory system and/or systemic cytokines. Consequences of neuronal infection include transcriptional dysregulation indicative of an inflammatory response (Jacob et al., 2020) , missorted and phosphorylated Tau (Ramani et al., 2020) , and increased cell death (Jacob et al., 2020; Ramani et al., 2020; Song et al., 2021) .

Large-scale international consortia have assembled to identify host-genetic associations with COVID-19 clinical outcomes (Initiative, 2020; Shelton et al., 2020; Tanigawa and Rivas, 2020) , although cohorts remain under-powered by genome-wide association study (GWAS) standards. To date, such studies have only found a handful of loci to be genome-wide significant (Initiative, 2020; Kachuri et al., 2020; Ramlall et al., 2020) . The largest data-freeze to date (https://www.covid19hg.org) does not include rs4702 within summary statistics; however, we note an excess of nominally significant genetic variants within the FURIN cis-region (Supplemental Table 1 ; binomial test p-value=3.57x10 -31 ) when comparing individuals hospitalized from COVID-19 to the general population (Initiative, 2020) . This indicates the necessity of expanding GWAS to more comprehensively explore the polygenic architecture for COVID-19 clinical outcome. Besides FURIN and components of the immune system, variants in ACE2, TMPRSS2 and BSG harbor genetic variants that are significantly enriched in COVID-19 patients and are predicted to influence susceptibility to SARS-CoV-2 infection (Benetti et al., 2020; Pairo-Castineira et al., 2020; Russo et al., 2020) . Such discoveries could help to generate hypotheses for drug repurposing, identify individuals at unusually high or low risk, and contribute to global knowledge of the biology of SARS-CoV-2 infection and disease. Discovery-based approaches to broadly identify all genes required for host infection and survival across tissues and cell types are also necessary to inform in silico drug repurposing screens (Han et al., 2021) . Integration of ongoing clinical GWAS with in vitro CRISPR-based forward genetic screening (e.g. Perturb-seq (Dixit et al., 2016) ) and pooled eQTL screens (e.g. crisprQTL mapping (Gasperini et al., 2019) and Census-seq (Mitchell et al., 2020) ) approaches will more rapidly identify host variants that impact the entry, replication and egress of SARS-CoV-2, cellular survival, and host immune response.

This study focused only on host genes known to mediate SARS-CoV-2 infection. The broad requirement of ACE2 and TMPRSS2 expression for SARS-CoV-2 infection across cell types was consistent with established literature , whereas the observed impact of BSG expression was more surprising given recent evidence suggesting that BSG is incapable of binding SARS-CoV-2 (Shilts et al., 2021) . Together with the data shown here, this suggests that BSG protein may enhance viral entry by means other than directly binding to or modifying SARS-CoV-2, that it may require a secondary cofactor, and/or that BSG has cell-type-specific functions in vivo. The impact of FURIN expression was not unexpected (Coutard et al., 2020; Wrapp et al., 2020) ; however, because rs4702 lies in the binding site for microRNA-338 (Hou et al., 2018) , this is a contextdependent eQTL, and so dependent upon cell-type-specific and donor-dependent expression of miR-338 (Schrode et al., 2019) . Overall, it remains possible that our in vitro findings will not predict clinical response because of the influence of other genetic and/or environmental factors. For example, SARS-CoV-2 antagonism of the immune response has been substantially defined (Konno et al., 2020; Lei et al., 2020; Thoms et al., 2020; Xia et al., 2020) , and so genetic variants that result in decreased replication efficiency of SARS-CoV-2 in vitro may ultimately prove advantageous for the virus in vivo, permitting low levels of virus to spread successfully while remaining undetected by the immune system.

The expected clinical response to SARS-CoV-2 infection in the donors from whom we derived hiPSCs is not known, therefore we cannot explore the extent to which in vitro findings recapitulate clinical outcomes. Moreover, hiPSC-derived cells of most (if not all) lineages are immature relative to those in adult humans (Hoffman et al., 2017; Jacob et al., 2019; Pavlovic et al., 2018) , which may impact SARS-CoV-2 infection/response, particularly since clinical outcomes in children are generally mild (Ludvigsson, 2020) . Given that clinical evidence suggests that SARS-CoV-2 infection can lead to heterogeneous acute and chronic outcomes across a range of tissues, future work should explore across the wider range of cell types, both the cell autonomous response to infection as well as noncell-autonomous effects resulting from subsequent cytokine storms that occur as part of response to infection. Moving forward, we should strive towards an expanded and unbiased isogenic strategy, that will prove more broadly useful in exploring the impact of host genome on SARS-CoV-2 infection across diverse cell types and tissues.

In summary, we demonstrate that a single non-coding SNP is sufficient to impact SARS-CoV-2 infection in human neurons and alveolar cells. This work supports ongoing efforts to discover host genes associated with SARS-CoV-2 infection, both in vitro and in the clinic. Our hope is that such efforts might better predict clinical outcomes before the onset of symptoms and facilitate the discovery of drugs that might prevent or treat COVID-19 disease.

Donors:

hiPSC lines and culture: Human induced pluripotent stem cells (hiPSCs) were cultured in StemFlex media (Gibco, #A3349401) and passaged with EDTA (Life Technologies #15575-020). All wildtype and shRNA experiments in lung alveolosphere cells, as well as all CRISPR-engineered AA and GG experiments were conducted in NSB3113, here C1 (female, 18 years old, European descent) and NSB3188, here C2 (female, 14 years old, European descent) (Schrode et al., 2019) . shRNA experiments in neurons were conducted in control NSB553 (male, 31 years old, European descent) and NSB2607 (male, 15 years old, European descent) hiPSC-derived NPCs (Hoffman et al., 2017) ; wildtype and shRNA intestinal cell experiments were conducted in 1383D6 (male, 36 years, Asian descent) (Takayama et al., 2017) .

SARS-CoV-2 virus propagation and infections: SARS-related coronavirus 2 (SARS-CoV-2), isolate USA-WA1/2020 (NR-52281) was deposited by the Center for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. SARS-CoV-2 was propagated in Vero E6 cells in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM Non-Essential Amino Acids, 1 mM Sodium Pyruvate and 10 mM HEPES. Virus stock was filtered by centrifugation using Amicon Ultra-15 Centrifugal filter unit (Sigma, Cat # UFC910096) and resuspended in viral propagation media. All infections were performed with either passage 3 or 4 SARS-CoV-2. Infectious titers of SARS-CoV-2 were determined by plaque assay in Vero E6 cells in Minimum Essential Media supplemented with 4 mM L-glutamine, 0.2% BSA, 10 mM HEPES and 0.12% NaHCO3 and 0.7% Oxoid agar (Cat #OXLP0028B). All SARS-CoV-2 infections were performed in the CDC/USDAapproved BSL-3 facility of the Global Health and Emerging Pathogens Institute at the Icahn School of Medicine at Mount Sinai in accordance with institutional biosafety requirements.

i. SARS-CoV-2 qPCR: Primers were used as Supplemental Experimental Procedures. The SARS-CoV-2 genome is comprised of two long ORFs that encode nonstructural polyproteins, followed by a series of largely structural genes. In order for these latter genes to be produced in the quantities necessary to manufacture mature virions, the replicase/transcriptase complex that is formed by the protein products of the long polyproteins recombine the "leader sequence," which initiates transcription with different lengths of the 3' end of the genome, generating "subgenomic" vRNA. The N primer sequences were designed so that the forward primer is specific to the leader sequence, and the reverse primer is specific to N. In this way, the primers should only amplify subgenomic vRNA, which should only be detected when the viral replication cycle has been initiated, as it is not present in packaged virions.

ii. Data analysis: Data from all phenotypic assays above were first organized in a Microsoft Excel spreadsheet and analyzed using GraphPad PRISM 8 software or R. For qPCR data analysis, values are expressed as mean ± SEM. Statistical significance was tested using either one-sided student Ttest or two-way ANOVA with Tukey's post-hoc test for comparison of all sample means.

All donor-derived and CRISPR-edited hiPSCs are available at the NIMH repository at RUCDR. Hamster brain SARS-CoV-2 RNA-sequencing datasets available at the Gene Expression Omnibus (GSE161200).

A. Expression of host genes associated with SARS-CoV-2 infection (Gordon et al., 2020; Ou et al., 2020; Wruck and Adjaye, 2020) examined by RNA-sequencing (Log 2 RNA-seq, CPM medians) of post-mortem human prefrontal cortex (PFC), human induced pluripotent stem cells (hiPSCs), neural progenitor cells (NPCs) and four hiPSC-derived neuronal cell types: induced glutamatergic (GLUT), differentiated forebrain (FB) populations (comprised of glutamatergic neurons, GABAergic neurons and astrocytes), induced GABAergic (GABA) and induced dopaminergic (DOPA). B. Immunoblot showing ACE2, FURIN and BSG protein in post-mortem human adult substantia nigra (SN) and PFC. C. Baseline host gene expression in d21 NGN2-induced glutamatergic neurons, uninfected or infected with SARS-CoV-2 (MOI 0.5), normalized relative to alveolospheres, to ensure comparability across cell types. Data are presented as mean ± SEM from two independent donors from two independent experiments, 1-2 replicates each. D. qPCR analysis of relative change in subgenomic vRNA N transcript and Interferon β (IFNB1) in mock and SARS-CoV-2 infected d21 NGN2-induced glutamatergic neurons (MOI of 0.05, 24 hours). Data are presented as mean ± SEM from two donors from two independent experiments, 1-2 replicates each. E. Representative immunostaining for SARS-CoV-2 nucleocapsid (N) protein (red), neuronal marker β-III-tubulin (green) and DAPI (blue) in d7 NGN2-induced glutamatergic neurons infected with a SARS-CoV-2 (MOI of 0.05, 48 hours). Scale bar: 50µm F. Immunoblot against SARS-CoV-2 nucleocapsid (N) protein shows replication in NGN2induced glutamatergic neurons at a MOI of 1 after 24 hours.

A. Expression of relevant host genes associated with SARS-CoV-2 infection in hamster brain after mock infection, 1 or 8 days following SARS-CoV-2 infection. Data are derived from at least two independent animals each. B. Volcano plot highlighting the top 50 significantly differentially expressed genes after a high pfu challenge with SARS-CoV-2. C. Interferon response over course of infection, expression shown in log2CPM, gene set from (Blanco-Melo et al., 2020) . D. Gene set enrichment for upregulated pathways using the MAGMA gene set. E. Gene set enrichment for downregulated pathways using curated gene sets (Schrode et al., 2019) .

A. Representative brightfield image of alveolosphere cultures on d30. 100µm. B. Representative immunofluorescence staining against surfactant protein C (SFTPC) and ACE2. Scale bar: 50µm. C. Baseline SARS-CoV-2 host gene expression in alveolospheres. Data are presented as mean ± SEM from two donors, from three independent experiments, with approximately 15 organoids per sample. D. Representative immunofluorescence staining against SARS-CoV-2 nucleocapsid (N) (red), epithelial marker EPCAM (green), NKX2-1 (blue, left) and DAPI (blue, right) in alveolospheres with mock or SARS-CoV-2 infection (MOI 0.5, 24 hours). Scale bar: 100µm. E. qPCR analysis of relative change in subgenomic vRNA N transcript in mock and SARS-CoV-2 infected d34 alveolospheres from two donors (MOI of 0.1 or 0.5, 24 hours). Data are presented as mean ± SEM from two donors, from three independent experiments, with approximately 15 organoids per sample. T-test, relative to respective mock, *p < 0.05, **p < 0.01. F. Dissociated alveolar cells after SARS-CoV-2 infection on day 34 are positive for SARS-CoV-2 N protein (red). Scale bar: 100µm.

A. Representative immunofluorescence staining of intestinal organoids for the intestinal markers CDX2 (red), KRT20 (green) and the SARS-CoV-2 host factor ACE2. Scale bar: 100µm. B. Immunofluorescence staining of intestinal organoids against SARS-CoV-2 nucleocapsid (N) (red), the epithelial marker EPCAM (green) and DAPI (blue), following mock or SARS-CoV-2 infection (MOI of 0.5 or 1, 48 hours). Scale bar: 100µm. C. Baseline SARS-CoV-2 host gene expression in mock treated intestinal organoids day 24 and d50. Data are presented as mean ± SEM from one donor, from two independent experiments, with approximately 15 organoids per sample. D. qPCR analysis of relative change in subgenomic vRNA N transcript in mock and SARS-CoV-2 infected intestinal organoids infected (MOI of 0.05 or 0.5, 24 hours). Data are presented as mean ± SEM from one donor, from two independent experiments, with approximately 15 organoids per sample.. T-test, relative to mock, *p < 0.05, **p < 0.01. E. Representative immunofluorescence staining of dissociated intestinal cells after SARS-CoV-2 infection on day 25 (MOI of 0.05, 48 hours), positive for SARS-CoV-2 N protein (red), EPCAM (green), DAPI (blue). Scale bar: 50µm. T-test, relative to scrambled shRNA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as mean ± SEM from one donor, from three independent experiments, 1-2 replicates each. (I.) Quantification of high-content imaging for SARS-CoV-2 nucleocapsid (N) protein in mock and SARS-CoV-2 infected dissociated intestinal cells infected at MOI of 0.05 and 0.5 for 48 hours. Each data point represents an independent well presented as mean, from two independent experiments. 2-way ANOVA, relative to respective scrambled shRNA control; ****p < 0.0001.

A. Representative immunofluorescence staining against SARS-CoV-2 nucleocapsid (N) protein (red), epithelial marker EPCAM (green), and DAPI (blue). Alveolospheres were generated from C2 FURIN rs4702 AA and GG lines and infected with mock or a MOI of 0.5 SARS-CoV-2 for 24 hours. CD163  CCR5  HELZ2  PARP9  IFIT3  IFIT2  IFNGR1  PARP12  TMEM140  TRIM25  RTP4  IFNLR1  IRF2  MYD88  IL6R  NMI  NLRC5  TRIM56  STAT2  NCOA7  PNPT1  CNP  IFNAR1  CXCL10  LAP3  GBP2  CXCL14  ADAR  SLC25A28  AXL  TDRD7  C1S  CCL8  OGFR  BST2  CSF1  IRF7  DHX58  IRF9  SP100  SP110  TRIM21 

Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Publicly Available hiPSC Lines with Extreme Polygenic Risk Scores for Modeling Schizophrenia

Genomewide Association Study of Severe Covid-19 with Respiratory Failure

Open Chromatin Profiling in hiPSC-Derived Neurons Prioritizes Functional Noncoding Psychiatric Risk Variants and Highlights Neurodevelopmental Loci

Gene expression elucidates functional impact of polygenic risk for schizophrenia

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Genetic effects on gene expression across human tissues

Clinical Characteristics of Coronavirus Disease 2019 in China

Identification of SARS-CoV-2 inhibitors using lung and colonic organoids

Neurologic Features in Severe SARS-CoV-2 Infection

Leveraging the antiviral type-I interferon system as a first line defense against SARS-CoV-2 pathogenicity

Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Schizophrenia-associated rs4702 G allele-specific downregulation of FURIN expression by miR-338-3p reduces BDNF production

SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

The COVID-19 Host Genetics Initiative, a global initiative to elucidate the role of host genetic factors in susceptibility and severity of the SARS-CoV-2 virus pandemic

Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium

The mutational constraint spectrum quantified from variation in 141,456 humans

Human Genetic Determinants of Viral Diseases

Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary

SARS-CoV-2 ORF3b Is a Potent Interferon Antagonist Whose Activity Is Increased by a Naturally Occurring Elongation Variant

SARS-CoV-2 productively infects human gut enterocytes

Activation and evasion of type I interferon responses by SARS-CoV-2

The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients

Human susceptibility and resistance to Norwalk virus infection

Systematic review of COVID-19 in children shows milder cases and a better prognosis than adults

Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease

Functional and cognitive outcomes after COVID-19 delirium

Mapping genetic effects on cellular phenotypes with "cell villages

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Genetic mechanisms of critical illness in Covid-19

A Comparative Assessment of Human and Chimpanzee iPSC-derived Cardiomyocytes with Primary Heart Tissues

Steroid-Responsive Encephalitis in Coronavirus Disease

SARS-CoV-2 targets neurons of 3D human brain organoids

Identification of Immune complement function as a determinant of adverse SARS-CoV-2 infection outcome. medRxiv

Genetic Analysis of the Coronavirus SARS-CoV-2 Host Protease TMPRSS2 in Different Populations

Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene

Synergistic effects of common schizophrenia risk variants

Trans-ethnic analysis reveals genetic and non-genetic associations with COVID-19 susceptibility and severity. medRxiv

No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor

Neuroinvasion of SARS-CoV-2 in human and mouse brain

Generation of safe and therapeutically effective human induced pluripotent stem cell-derived hepatocyte-like cells for regenerative medicine

Initial Review and Analysis of COVID-19 Host Genetics and Associated Phenotypes

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

Guillain-Barre Syndrome Associated with SARS-CoV-2

NCBI's Database of Genotypes and Phenotypes: dbGaP

Presence of Genetic Variants Among Young Men With Severe COVID-19

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell

CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

SARS-CoV-2 receptor ACE2 is co-expressed with genes related to transmembrane serine proteases, viral entry, immunity and cellular stress

Evasion of Type I Interferon by SARS-CoV-2

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids

Generation of pure GABAergic neurons by transcription factor programming

A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV

Inborn errors of type I IFN immunity in patients with lifethreatening COVID-19

Human iPSC-Derived Posterior Gut Progenitors Are Expandable and Capable of Forming Gut and Liver Organoids

Rapid single-step induction of functional neurons from human pluripotent stem cells

Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med. Highlights • hiPSC-derived model of SARS-CoV-2 infection in human neurons

• Infection of post-mitotic human neurons by SARS-CoV-2

Effect of host gene expression (ACE2, FURIN, BSG, and TMPRSS2) on SARS-CoV-2 infection

• Impact of host genotype, common single nuclear polymorphism rs4702

Akbarian and colleagues combine human induced pluripotent stem cell (hiPSC)-based models and CRISPR-engineering to explore the host genetics of SARS-CoV-2 in alveolar cells, intestinal cells, and neurons. A single nucleotide polymorphism (rs4702) located in the 3'UTR of the protease FURIN impacts alveolar and neuron infection by SARS-CoV-2 in vitro