key: cord-0877269-qinkcpbg
authors: Beumer, Joep; Geurts, Maarten H.; Lamers, Mart M.; Puschhof, Jens; Zhang, Jingshu; van der Vaart, Jelte; Mykytyn, Anna Z.; Breugem, Tim I.; Riesebosch, Samra; Schipper, Debby; van den Doel, Petra B.; Lau, Wim de; Pleguezuelos-Manzano, Cayetano; Busslinger, Georg; Haagmans, Bart L.; Clevers, Hans
title: A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses
date: 2021-05-20
journal: bioRxiv
DOI: 10.1101/2021.05.20.444952
sha: 1689b2e8c57bcf23a7bedf41b34b5cdfacff6869
doc_id: 877269
cord_uid: qinkcpbg

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 (IOs) 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.

immunohistochemistry (Fig. S7A) . These experiments were performed using a VeroE6-propagated 158 stock and recent work has pointed out that propagation on VeroE6 cells can lead to culture adaptive 159 mutations in the multibasic cleavage site [39] [40] [41] [42] [43] . The VeroE6 stock used in this study was deep-160 sequenced 39 and was 64.2% wild-type in the RRAR (spike positions 682-685) multibasic cleavage site. 161 We detected another mutation adjacent to the multibasic cleavage site (S686G) at a frequency of 162 45.4%. Viruses with multibasic cleavage site cleavage site mutations, including S686G, were shown to 163 slightly increase cathepsin usage by ~20% 39 , indicating that the majority of these viruses still used 164 serine protease-mediated entry. 165 We have previously shown that propagation in TMPRSS2-expessing Calu-3 cells prevents culture 166 adaptation. Using this Calu-3 stock that was completely non-adapted 39 , we confirmed the dependency 167 of SARS-CoV-2 on TMPRSS2 (and ACE2) (Fig. 4C ). Immunofluorescence of TMPRSS2-deficient organoids 168 showed absence of viral spread (Fig. 4D ). This implied that TMPRSS2 is the main proteolytic activator 169 of the SARS-CoV-2 spike protein. In contrast to knockout screens in VeroE6 cells that showed that the 170 endocytic pathway protease Cathepsin L was essential for SARS-CoV-2 entry 9 , SARS-CoV-2 replicated -171 if anything-more efficiently in Cathepsin L-mutant than in wildtype organoids ( Fig. 4A-B) . Efficient 172 depletion of Cathepsin L was supported using western blot analysis (Fig. S7B) . We confirmed the 173 obligate role of TMPRSS2 for SARS-CoV-2 replication in IOs derived from a different donor and from 174 another segment (duodenum) of the human intestine (Fig. 4E) . 175

Since we observed differential expression of multiple proteases -including upregulation of cathepsins 176 -in differentiated organoids, we additionally assessed TMPRSS2-dependency in differentiated 177 intestinal cells (Fig. S7C ). After 5 days of differentiation, both wildtype and TMPRSS2-mutant organoids 178 were infected with SARS-CoV-2. SARS-CoV-2 replicated efficiently in differentiated organoids, as we 179 reported previously 17 . Viral replication was greatly diminished in TMPRSS2-deficient organoids, 180 suggesting dependency on this protease across different intestinal cell types (Fig. S7D ) 181

To assess redundancy in single TMPRSS-or cathepsin-mutant organoids, we additionally generated 182 organoids mutant for both CTSL/CTSB, or TMPRSS2/4, the most abundantly expressed cathepsins and 183 serine proteases in the intestinal epithelium. Previous work implied a role for TMPRSS4 in viral entry 184 in the intestinal epithelium 33 . We did not observe reduced replication when both cathepsins were lost. 185

Moreover, TMPRSS4 knock-out in a TMPRSS2-mutant background did not further decrease infectivity 186 (Fig. S7E ). In line with this, the broad serine protease inhibitor camostat did not affect replication in 187 TMPRSS2-deficient IOs (Fig. S7F ). We concluded that the cathepsins and TMPRSS4 do not play a role 188 in viral entry in the intestinal epithelium. 189

To confirm that the endocytic pathway is dispensable for viral entry, we treated IOs with 1) the 190 endosomal pathway inhibitor chloroquine, the cathepsin protease inhibitor E64D, or the broad serine 191 protease inhibitor camostat. These drugs were well-tolerated with no growth impairment at the 192 concentrations used (Fig. S8A-B) . As published previously, chloroquine was effective in VeroE6 cells 193 (Fig. S8C) 7 . While camostat effectively inhibited viral replication, chloroquine and E64D did not affect 194 replication in IOs (Fig. 4F ). E64D-treated organoids displayed a trend towards more efficient viral 195 replication (Fig. 4F) , consistent with observations in the Cathepsin L-mutant organoids (Fig. 4A-B) . We 196 concluded that Cathepsin-mediated entry through the endosomal route may be the central port of 197 viral entry in cell lines, but not in IOs, in which SARS-CoV-2 enters through the activity of TMPRSS2 (Fig  198   4A -B). These observations may also explain why (hydro)-chloroquine has emerged from cell line 199 studies but has proven ineffective in the clinic. 200

We further tested IOs mutant in a range of non-protease host factors to assess their role in coronavirus 202 replication, of which some have already been linked to the SARS-CoV-2 replication cycle. NRP1 recently 203 attracted attention as a novel co-receptor for SARS-CoV-2 in two separate studies that used Hela, 204 HEK293T and the colorectal cancer cell line Caco-2 13,14 . These findings were substantiated by x-ray 205 crystallography data supporting binding of the viral spike protein to NRP1 SARS-CoV-2 infection was 206 significantly inhibited by NRP1-blocking antibodies 13,14 . Additionally, CD209 was recently identified as 207 potential SARS-CoV-2 receptor, and facilitated viral entry in HEK-293 cells when overexpressed 36 . A 208 recent study found that SARS-CoV-2 can bind heparan sulfate on the cell surface through its spike 209 protein. When enzymes involved in the sulfation of heparan sulfate, including NDST1, were knocked 210 out in Hep3B cells, viral replication was almost entirely abolished 12 . None of these, nor the additional 211 host factors we assessed, significantly impacted on viral replication when mutated in IOs (Fig. 5A, Fig.  212 S9). We conclude that all of these proteins would therefore not be viable drug targets for the treatment 213 of COVID-19 (Fig. 5B ). Further studies may assess whether loss of these factors influence the cellular 214 response to coronaviruses in any other way than replication efficiency. 215

The mutant host factor KO biobank can readily be employed when new coronaviruses or viral strains 217 appear, to assess the dependency on host factors and identify druggable targets. We first tested 218 whether the same TMPRSS2-dependency exists for the other two coronaviruses. SARS-CoV replication 219 was strongly diminished upon TMPRSS2 loss, while MERS-CoV replication was reduced more modestly 220 (Fig. 6A) . The latter potential redundancy may be explained by the presence of two functional 221 multibasic cleavage sites in the MERS-CoV spike, whereas SARS-CoV-2 and SARS-CoV possess one and 222 none, respectively 22 . Both viruses could replicate in the absence of Cathepsin L, suggesting that 223 coronaviruses generally do not use the endosomal entry route in primary epithelial cells as present in 224 organoids (Fig. 6A) . 225

Recently, a novel SARS-CoV-2 variant (clade B.1.1.7 or British variant) emerged and is rapidly replacing 226 endemic viruses globally. Epidemiological data suggest that this variant is 1.35-2 fold more 227 transmissible than the ancestral lineage and is associated with higher viral loads 44-46 . Interestingly, this 228 variant contains a mutation (P681H) directly N-terminal from the RRAR multibasic cleavage site that 229 adds another basic residue to the multibasic cleavage site, creating an HRRAR motif. A similar mutation 230 (P681R) was detected in the Indian variant (clade B.1.617). As the multibasic cleavage site facilitates 231 serine protease-mediated entry 11 , mutations in or near this site may alter protease usage for S2' 232 cleavage, which directly triggers fusion and entry. We found that the British variant replicated 233 efficiently in wildtype and cathepsin mutant organoids, but not in TMPRSS2-deficient cells (Fig. 4E) , 234

indicating that the British variant did not broaden its protease usage. These experiments provide a 235 proof-of-concept on how emerging viral strains could be screened against mutant IOs. 236

The current COVID-19 pandemic has exposed weaknesses in our preparedness for coronavirus 238 pandemics. No effective coronavirus antivirals are approved for use in humans and all completed large-239 scale COVID-19 drug trials have failed to show efficacy to this date, including (hydroxy)chloroquine and 240 remdesivir 8,47 . The disappointing clinical effects of (hydroxy)chloroquine in humans in particular 241 highlights gaps in the understanding of fundamental coronavirus biology. (Hydroxy)chloroquine, an 242 inhibitor of the endosomal acidification was identified as a potent inhibitor of SARS-CoV 48 and SARS-243

CoV-2 7 viral entry in cell line-based assay, confirmed here. In agreement with this, recent whole 244 genome CRISPR/Cas9 genetic screens in transformed cell lines again suggested that endosomal entry 245 factors, such as cathepsin L, are crucial for SARS-CoV-2 entry 9,49,50 . 246

Here, we use human intestinal organoids as a non-transformed model to study genes implicated in 247 coronavirus biology. We have chosen to use only IOs since it is currently not possible to efficiently 248 genetically engineer airway organoids due to limited clonal outgrowth of these cells. Nevertheless, IOs 249 express the majority of host factors assessed, including proteases, to a similar level as the airways (Fig.  250 1). We confirmed that in this model ACE2 is the obligate entry receptor for SARS-CoV-2 and SARS-CoV, 251 while DPP4 is the entry receptor for MERS-CoV, indicating that accessory receptors may not play crucial 252 roles for these viruses. Indeed, knockout of NRP1, recently proposed as a SARS-CoV-2 (co-)receptor in Cathepsin L and B are not involved in SARS-CoV-2 entry in IOs. In accordance with this, a cathepsin 255 inhibitor (E64D) and chloroquine did not inhibit SARS-CoV-2 in these IOs, while the serine protease 256 inhibitor Camostat effectively blocked viral propagation. A similar anti-SARS-CoV-2 effect of camostat 257 was observed in organoid-derived airway cells 11 . The broad activity of Camostat does not allow to 258 pinpoint which serine protease mediates entry. 259 TMPRSS2-deficiency in IOs strongly decreased SARS-CoV-2 replication and spread, indicating that 260 TMPRSS2 is the main priming protease. Other related TMPRSS genes have previously also been linked 261 to SARS-CoV-2 replication, including TMPRSS4 in the intestine 33 Altogether, these findings indicate that multiple TMPRSS genes may be able to mediate entry when 273 overexpressed, but -at physiological levels in IOs-only TMPRSS2 plays an essential role, which may 274 inspire the development of high-specificity TMPRSS2 inhibitors. The high TMPRSS-2 dependency of 275 SARS-CoV (this study) indicates that such inhibitors may well be effective against future SARS-like 276 coronavirus pandemics. The observation that TMPRSS2-null mice do not display a visible phenotype 53 277 implies that such inhibitors may be well-tolerated. Our findings match with observations that SARS-278

CoV and to a lesser extent MERS-CoV replication and dissemination was reduced in TMPRSS2-deficient 279 mice 54 . 280

In conclusion, our findings underscore the relevance of non-transformed human models for 281 (corona)virus research and identify TMPRSS2 as an attractive therapeutic target in contrast to many 282 other genes (e.g. cathepsin L, cathepsin B, NRP1, NDST1 etc) that -as deduced from our observations-283 unlikely to be of clinical value. Future emerging viruses could be readily screened against our IO host 284 factor knockout biobank to rapidly identify therapeutic targets.

We thank Single Cell Discoveries for RNA library preparation, and R. van der Linden and S. van Elst for 287 help with FACS sorting. We thank E. Eenjes and R. Rottier for providing human lung material. 288 Isolation of human bronchial airway stem cells was performed using a protocol similar to Sachs and 485 colleagues 57 . Small airway stem cells were isolated from distal human lung parenchyma as described 486 before 57 . Tracheal stem cells were collected from tracheal aspirates of intubated preterm infants (28 487 weeks gestational age). Organoids were cultured as described before 57 . To obtain differentiated 488 organoid-derived cultures, organoids were dissociated into single cells using TrypLE express (Gibco; 489 #12604013). Cells were seeded on Transwell membranes (Corning) coated with rat tail collagen type I 490 (Fisher Scientific). Single cells were seeded in AO growth medium : complete base medium (CBM; 491 Stemcell Pneumacult-ALI; #05001) at a 1:1 ratio. After 2-4 days, confluent monolayers were cultured 492 at air-liquid interphase in CBM. Medium was changed every 5 days for 8 weeks. 493

sgRNAs targeting loci of interest were cloned into a SpCas9-EGFP vector (addgene plasmid #48138) 495 using a protocol described before 58 . sgRNAs were designed using WTSI website 496 (https://www.sanger.ac.uk/htgt/wge/). A full list of gRNAs and primers to generate SpCas9-EGFP 497 expressing plasmids can be found in supplementary table 5. To generate homozygous frameshift 498 mutations in genes of interest, organoids were transfected with SpCas9-EGFP containing the locus-499 specific sgRNA. Transient transfection using a NEPA21 electroporator was performed as described 500 before 59 . 3-7 days after transfection, organoids were dissociated using TryplE (TryplE Express; Life 501 Technologies) and sorted on a FACS-ARIA (BD Biosciences) for GFP positivity. After sorting, Rho kinase 502 inhibitor (Y-27632 dihydrochloride; 10µM, Abmole) was added for 1 week to support single cell 503 outgrowth. 504

To generate clonal organoid lines with genotypes of preference, organoids were picked 2 weeks after 506 sorting. Manually picked organoids were dissociated using TryplE (TryplE Express; Life Technologies) 507 and plated in BME in pre-warmed cell culture plates. After two weeks, single cells grew into organoids 508 and were split again to verify actively dividing stem cells. After the second split, 20µL of organoid-BME 509 suspension was directly taken from the plate and DNA was extracted from the organoids using the 510 Zymogen Quick-DNA microprep kit according to protocol. Regions around sgRNA target sites were 511 amplified using Q5 high fidelity polymerase (NEB) according to manufacturer's protocol. CRISPR/Cas9-512 mediated indel formation was confirmed by sanger sequencing of these amplicons (Macrogen). 

Infections were performed using a protocol similar to 17 . Briefly, organoids were harvested in cold 541

Advanced DMEM (including HEPES, Glutamax and antibiotics, termed AdDF+++ 17 ), washed once in 542 AdDF+++, and sheared using a flamed Pasteur pipette in AdDF+++. Differentiated organoids were 543 broken using a 5-minute incubation with TrypLE (TrypLE Express; Life Technologies). After shearing, 544 organoids were washed once in AdDF+++ before infection was performed in expansion medium. For 545 the experiment in Figure S5B , organoids were gently harvested using a wide pipet tip to avoid shearing 546 organoids. A multiplicity of infection (MOI) of 0.1 was used for SARS-CoV and SARS-CoV-2 and an MOI 547 of 0.2 was used for MERS-CoV. After 2 hours of virus adsorption at 37°C 5% CO2, cultures were washed 548 four times with 4 ml AdDF+++ to remove unbound virus. Organoids were re-embedded into 30 μL BME 549 in 48-well tissue culture plates and cultured in 250 μL expansion or differentiation medium at 37°C 550 with 5% CO2. Each well contained ~200,000 cells per well. At indicated time points cells were harvested 551 by resuspending the BME droplet containing organoids into 200 μL AdDF+++. Samples were stored at 552 -80°C, a process which lysed the organoids, releasing their contents into the medium upon thawing. 553

For testing the antiviral activity of chloroquine diphosphate (Sigma), camostat mesylate (Sigma) and 554 E64D (Medchemexpress) in intestinal organoids, sheared organoids were preincubated with these 555 compounds in AdDF+++ at the indicated concentrations for 30 min at 37°C 5% CO2 before infection at 556 an MOI of 0.1 with SARS-CoV-2. After virus adsorption for 2 hours at 37°C 5% CO2, organoids were 557 washed and re-embedded in BME as indicated above. Medium containing the inhibitors was added to 558 the wells for the duration of the experiment. Cells were harvested at indicated time points as described 559

above and stored at -80°C. 560

Chloroquine was two-fold serially diluted in Opti-MEM I (1X) + GlutaMAX starting from a concentration 562 of 100 µg/mL. 100 μl of each dilution was added to a confluent 96-well plate of VeroE6 cells and pre-563 incubated at 37°C 5% CO2 for 30 minutes. Next, cells were incubated with 400 plaque-forming units of 564 virus in the same concentration range of chloroquine at 37°C 5% CO2. After 8 hours incubation, cells 565

were fixed with formalin, permeabilized with 70% ethanol and stained with polyclonal rabbit anti-566 SARS-CoV nucleoprotein antibody (1:1000; 40588-T62, Sino Biological) followed by secondary 567

Alexa488 conjugated goat-anti-rabbit antibody (Invitrogen). Plates were scanned on the Amersham™ 568 Typhoon™ Biomolecular Imager (channel Cy2; resolution 10µm; GE Healthcare). Data was analyzed 569 using ImageQuant TL 8.2 image analysis software (GE Healthcare). 570

For determining the viral titer using qPCR, samples were thawed and centrifuged at 2,000 g for 5 min. 572

Sixty μL supernatant was lysed in 90 μL MagnaPure LC Lysis buffer (Roche) at RT for 10 min. RNA was 573 extracted by incubating samples with 50 μL Agencourt AMPure XP beads (Beckman Coulter) for 15 min 574 at RT, washing beads twice with 70% ethanol on a DynaMag-96 magnet (Invitrogen) and eluting in 30 575 μL MagnaPure LC elution buffer (Roche). Viral titers (TCID50 equivalents per mL) were determined by 576 qRT-PCR using primer-probe sets described previously 60-62 and comparing the Ct values to a standard 577 curve derived from a titrated virus stock. 578

Organoids were stained as described before 55 . Whole organoids were collected by gently dissolving 580 the BME in ice-cold PBS, and subsequently fixed overnight at 4°C in 4% paraformaldehyde (Sigma). 581

Next, organoids were permeabilized and blocked in PBS containing 0,5% Triton X-100 (Sigma) and 2% 582 normal donkey serum (Jackson ImunoResearch) for 30 min at room temperature. All stainings were 583 Alexa488-, 568-and 647-conjugated anti-rabbit and anti-goat (1:1,000; Molecular Probes) or 589

Phalloidin-Alexa488 (Thermofisher Scientific) in blocking buffer containing 4ʹ,6-diamidino-2-590 phenylindole (DAPI; 1;1,000, Invitrogen). After staining, organoids were transfected to a glass slide 591 embedded in Vectashield (Vector labs). Stained organoids were imaged using a SP8 confocal 592 microscope (Leica) or a Zeiss LSM700, and image analysis and presentation was performed using 593 ImageJ software. 594 Immunohistochemistry was performed as described before 63 . Antigen retrieval for TMPRSS2 staining 595 was achieved by boiling for 20 minutes in citrate buffer. Primary antibody used was rabbit anti-596 TMPRSS2 (1:100; Abcam, ab109131) followed by anti-rabbit conjugated to horseradish peroxidase 597 (Powervision, Leica) For Western blot of CTSL, organoid proteins were solubilized using a standard RIPA buffer for 30 min 599 on ice in the presence of protease inhibitors. Samples were run on a 4-15% PAA gel (BioRad) under 600 reducing conditions. Proteins were electrophoresed to PVDF membranes from Immobilon.Both 601 primary antibodies, mouse anti-CTSL (± 25 kDa) and mouse anti-ITGB4 (± 200 kDa), were incubated 602 O/N at 4C in PBS/10% milk protein/0.1% Tween20. The secondary rabbit anti-mouse HRP-conjugate 603 TMPRSS2  TMPRSS3  TMPRSS4  TMPRSS5  TMPRSS7  TMPRRS11A  TMRRSS11D  TMPRSS11E  TMPRSS13  CTSL  CTSB   ACE2  DPP4  ANPEP  CD9  CEACAM1  NDST1  CLEC2B  FURIN  HSPA5  MAVS  VIM 

Clone 2 C11orf96  SLC34A2  IFIT2  DKK1  WFDC21P  CD274  LINC00941  RRAD  SNAI1  CYR61  NPTXR  ARC  FAAHP1  IFIT1  FOSB  C10orf99  LINC00543  ADH1C  LOC105377924  CA1  RN7SK   NBPF22P  HSPA6  CRYAB  LBH  C11orf96  SLC34A2  IFIT2  DKK1  WFDC21P  CD274  LINC00941  RRAD  SNAI1  CYR61  NPTXR  ARC  FAAHP1  IFIT1  FOSB  C10orf99  LINC00543  ADH1C  LOC105377924  CA1  RN7SK TOP AFTER 24 HOURS SARS-CoV-2 

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