A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

Abstract

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.

SciScore for 10.1101/2021.05.20.444952: (What is this?)

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

Table 1: Rigor

Ethics	Consent: Cell culture of human intestinal organoids and human airway: Human small intestinal tissue was obtained from the UMC Utrecht with informed consent of the patient. IACUC: The study was approved by the UMC Utrecht (Utrecht, The Netherlands) ethical committee and was in accordance with the Declaration of Helsinki and according to Dutch law. IRB: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512).
Sex as a biological variable	not detected.
Randomization	The experiments were not randomized and the investigators were not blinded to the sample allocation during experiments and outcome assessment.
Blinding	The experiments were not randomized and the investigators were not blinded to the sample allocation during experiments and outcome assessment.
Power Analysis	Quantification and statistics: No statistical methods were used to predetermine sample size.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After 8 hours incubation, cells were fixed with formalin, permeabilized with 70% ethanol and stained with polyclonal rabbit anti-SARS-CoV nucleoprotein antibody (1:1000; 40588-T62, Sino Biological) followed by secondary Alexa488 conjugated goat-anti-rabbit antibody (Invitrogen).	polyclonal rabbit suggested: None antibody ( Invitrogen) . suggested: None
For immunofluorescence, primary antibodies used were mouse anti-nucleoprotein (1:200; 40143-MM05, Sino Biological),	anti-nucleoprotein suggested: (Sino Biological Cat# 40143-MM05, RRID:AB_2827977)
mouse anti-dsRNA (1:200; Scicons), goat anti-ACE (1:100; R&D Systems, AF933), goat anti-DPP-4 (1:200; R&D systems, AF1180) and rabbit anti-MERS S1 (1:200; 40069-T52, Sino Biological).	anti-ACE suggested: (LSBio (LifeSpan Cat# LS-C96272-200, RRID:AB_2032850)
For immunofluorescence, organoids were incubated with the corresponding secondary antibodies Alexa488-, 568- and 647-conjugated anti-rabbit and anti-goat (1:1,000; Molecular Probes) or Phalloidin-Alexa488 (Thermofisher Scientific) in blocking buffer containing 4ʹ,6-diamidino-2-phenylindole (DAPI; 1;1,000, Invitrogen).	anti-rabbit suggested: None anti-goat suggested: None
Primary antibody used was rabbit anti-TMPRSS2 (1:100; Abcam, ab109131) followed by anti-rabbit conjugated to horseradish peroxidase (Powervision, Leica) For Western blot of CTSL, organoid proteins were solubilized using a standard RIPA buffer for 30 min on ice in the presence of protease inhibitors.	anti-TMPRSS2 suggested: (Abcam Cat# ab109131, RRID:AB_10863728)
Both primary antibodies, mouse anti-CTSL (± 25 kDa) and mouse anti-ITGB4 (± 200 kDa), were incubated O/N at 4C in PBS/10% milk protein/0.1% Tween20.	anti-CTSL suggested: None anti-ITGB4 suggested: (LSBio (LifeSpan Cat# LS-C43630-200, RRID:AB_1055018)
The mouse IgG2a antibody against ITGB4, 58XB4, was a gift from A. Sonnenberg (NKI, Amsterdam, The Netherlands).	mouse IgG2a suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Viruses and cell lines: Vero and VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS), HEPES, sodium bicabonate, penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.	Vero suggested: None
Calu-3 cells were maintained in Opti-MEM I (1X) + GlutaMAX (Gibco)(Gibco) supplemented with 10% FCS, penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.	Calu-3 suggested: None
SARS-CoV (isolate HKU 39849, genbank accession no. AY278491), SARS-CoV-2 (isolate Bavpat-1; European Virus Archive Global #026V-03883; kindly provided by Dr. C. Drosten) were propagated on VeroE6 cells in Opti-MEM I (1X) + GlutaMAX (Gibco), supplemented with penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
Recombinant DNA
Sentences	Resources
Transfection of organoids for CRISPR-Cas9 experiments: sgRNAs targeting loci of interest were cloned into a SpCas9-EGFP vector (addgene plasmid #48138) using a protocol described before58. sgRNAs were designed using WTSI website (https://www.sanger.ac.uk/htgt/wge/).	SpCas9-EGFP suggested: None
Software and Algorithms
Sentences	Resources
To obtain differentiated organoid-derived cultures, organoids were dissociated into single cells using TrypLE express (Gibco; #12604013).	Gibco suggested: None
CRISPR/Cas9-mediated indel formation was confirmed by sanger sequencing of these amplicons (Macrogen).	Macrogen suggested: (Macrogen, RRID:SCR_014454)
Data was analyzed using ImageQuant TL 8.2 image analysis software (GE Healthcare).	ImageQuant suggested: (ImageQuant, RRID:SCR_014246)
Stained organoids were imaged using a SP8 confocal microscope (Leica) or a Zeiss LSM700, and image analysis and presentation was performed using ImageJ software.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
Read 2 was aligned to the CRCh38 human RefSeq transcriptome, with the addition SARS-CoV-2 (Ref-SKU: 026V-03883) or MERS (NC_038294.1) genomes, using BWA using standard settings64.	BWA suggested: (BWA, RRID:SCR_010910)
Normalization using the median of ratios method and differential gene expression analysis was performed using the DESeq2 package64.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
Statistical analysis was performed with the GraphPad Prism 9 software.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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:

The current COVID-19 pandemic has exposed weaknesses in our preparedness for coronavirus pandemics. No effective coronavirus antivirals are approved for use in humans and all completed large-scale COVID-19 drug trials have failed to show efficacy to this date, including (hydroxy)chloroquine and remdesivir8,47. The disappointing clinical effects of (hydroxy)chloroquine in humans in particular highlights gaps in the understanding of fundamental coronavirus biology. (Hydroxy)chloroquine, an inhibitor of the endosomal acidification was identified as a potent inhibitor of SARS-CoV48 and SARS-CoV-27 viral entry in cell line-based assay, confirmed here. In agreement with this, recent whole genome CRISPR/Cas9 genetic screens in transformed cell lines again suggested that endosomal entry factors, such as cathepsin L, are crucial for SARS-CoV-2 entry9,49,50. Here, we use human intestinal organoids as a non-transformed model to study genes implicated in coronavirus biology. We have chosen to use only IOs since it is currently not possible to efficiently genetically engineer airway organoids due to limited clonal outgrowth of these cells. Nevertheless, IOs express the majority of host factors assessed, including proteases, to a similar level as the airways (Fig. 1). We confirmed that in this model ACE2 is the obligate entry receptor for SARS-CoV-2 and SARS-CoV, while DPP4 is the entry receptor for MERS-CoV, indicating that accessory receptors may not play crucial roles for these viruses....

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

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