Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Full length ACE2 was optimized to fit screening. Synthetic signal sequence and HA tag were fused to mature ACE2 and restriction sites were introduced by optimizing codon optimization for the mutated fragment replacement. ( B ) ACE2 mutant library was expressed in 293T cells and incubated with the RBD of SARS-CoV-2 fused to superfolder GFP (sfGFP). ( C ) Error-prone PCR amplification of ACE2 protease domain induced random mutations. Mutant library-transduced cells were incubated with the RBD-sfGFP. Top 0.05 % population with high level of bound RBD-sfGFP was sorted and underwent DNA extraction, followed by next cycle mutagenesis. ( D ) PD1 mutagenesis and high affinity selection were performed 3 times and followed by PD2 random mutation. Top 0.05% population were harvested and reconstructed into the backbone plasmid to be verified individually. ( E ) Neutralization activity against the RBD was evaluated with sACE2-sfGFP and the RBD-sfGFP. Serial dilution of sACE2-sfGFP was analyzed with the 50-fold diluted RBD-sfGFP in flow cytometry. The experiments were independently performed twice and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Sequencing, Mutagenesis, Incubation, Amplification, DNA Extraction, Selection, Plasmid Preparation, Neutralization, Activity Assay, Serial Dilution, Flow Cytometry

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Table of all ACE2 variants with mutations. ( B ) Kinetic analysis of sACE2-His binding to RBD-Fc was analyzed by surface plasmon resonance (SPR). ( C ) Neutralization potency of sACE2-Fc against SARS-CoV-2 or SARS-CoV-1 pseudotyped lentivirus was measured in 293T/ACE2 cells. Data are mean ± SD of n = 3 biological replicates. ( D ) Neutralization potency to authentic SARS-CoV-2 was analyzed in Vero6E/TMPRSS2 cells. Data are mean of n = 3 technical replicates.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Binding Assay, SPR Assay, Neutralization

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: An average mass of spike trimer was measured to be ∼560 kDa (top panel). All ACE2 molecules added in excess to the mixture samples gave a peak with an average mass of ∼90 kDa. The spike protein complexed with WT ACE2 showed a broad mass distribution between 550∼850 kDa (the second panel). In contrast, the spike protein complexed with the 3N39 or 3N39v2 showed a monodisperse distribution with an average mass of ∼830 kDa (the third and bottom panels), which corresponds to the mass of the spike trimer bound by three ACE2 molecules.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques:

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: Various versions of ACE2-His proteins were subjected to the differential scanning fluorimetry using SYPRO™ Orange as the probe dye. Denaturation curves were replotted for - d F/ d T and the peak temperature was estimated to be the Tm for each mutant.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: Neutralizing activity of ACE2-Fcs against soluble spike trimer binding to cell-surface ACE2 was evaluated in flow cytometry. Indicated concentration of spike trimer was incubated with 60 µg/ml (∼315 nM) ACE2-Fc proteins for 2h and then the mixture was reacted with ACE2-expressing Expi293F cells. Although WT ACE2-Fc can block binding of soluble spike protein to cells when the concentration of the spike was 0.3 nM, it could not outcompete spike at higher concentration. In contrast, complete (for 3N39v2 and J320v2) or near-complete (for J113v2) inhibition against 30 nM spike protein was achieved with the ACE2 mutants we have isolated, indicating the >100-fold increase in the blocking efficacy from the WT. The experiments were independently performed twice and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Activity Assay, Binding Assay, Flow Cytometry, Concentration Assay, Incubation, Expressing, Blocking Assay, Inhibition, Isolation

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Overall structure. 3N39 ACE2 and RBD are shown in orange and green, respectively. The mutated residues in 3N39 are shown as cyan stick models. The expanded views of the PD1 region are provided in the inset. ( B ) Structural comparison of the K31N/E35K mutation site in 3N39 (left panel) with its corresponding site in WT (right panel). Hydrogen-bonding interactions (within 3.0 Å) are indicated by dashed lines. ( C ) Structural comparison of the L79F/A25V mutation site in 3N39 (left panel) with its corresponding site in WT (right panel). F486 residue of RBD and hydrophobic residues composing the F486 binding pocket of ACE2 are shown as stick models with transparent sphere models.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Surface representations of the ACE2 structures. All ACE2 molecules are viewed from the same orientation (side view). The RBD-bound 3N39 (left) and the inhibitor-bound WT (center, 1r4l) adopt a closed conformation, while the RBD-bound WT (right, 6m0j) adopts an open conformation. ( B and C ) Superposition of the RBD-bound 3N39 (orange) and the inhibitor-bound WT (cyan, 1r4l) structures. Overall view and the expanded view of the enzymatic active site are provided in (B) and (C), respectively. The active site Zn ions (in both structures) are shown as sphere models, and a sulfate ion (in 3N39) and the inhibitor MLN-4760 (in 1r4l) are shown as stick models. ( D ) Comparison of the distances between C β atoms of S128 and V343 residues in the closed and open conformations. ( E ) SDS-PAGE analysis of WT and S128C/V343C mutant ACE2-His samples conducted under non-reducing (NR) and reducing (R) conditions. ( F ) Enzyme activity was assayed in the form of soluble ACE2-sfGFP by measuring Mca fluorescence resulting from hydrolysis of Mca-Ala-Pro-Lys(Dnp)-OH. ( G ) The RBD neutralization assay was performed in S128C/V343C mutant. The experiments were independently performed twice and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: SDS Page, Mutagenesis, Activity Assay, Fluorescence, Neutralization

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: ( A ) Protocol of generating escape mutation in SARS-CoV-2. At first, 0.1 MOI of SARS-CoV-2 was cultured in Vero6E/TMPRSS2 cells with indicated concentration of ACE2-Fc or H4 antibody, then a total of 3 × 10 5 copies of virus in partially neutralized well was passed in the presence of ACE2-Fc or H4 antibody dilution. Supernatants were collected from each well and analyzed virus RNA copy number by quantitative PCR. ( B ) The copy number of SARS-CoV-2 genome RNA in cultured medium was analyzed in each passage. The virus from indicated well (arrow head) was passed and escape mutant expansion was observed only in H4 antibody at passage 4. The test for 3J113v2 was discontinued due to no growth of virus at passage 2.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Mutagenesis, Cell Culture, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: High affinity modified ACE2 receptors protect from SARS-CoV-2 infection in hamsters

doi: 10.1101/2020.09.16.299891

Figure Lengend Snippet: Western blot of cultured medium from each ACE2-Fc transfected 293T cells. The experiments were independently performed 3 times and similar results were obtained. One representative data were shown.

Article Snippet: The plenti ACE2 vector was digested with BamBI or MfeI-SacII (NEB) for ACE2 residues 18-102 or 272-409, respectively with alkaline phosphatase (Fermentas) at 37 °C for 2 hr and gel-purified on a Gel and PCR Clean-up kit (TAKARA).

Techniques: Western Blot, Cell Culture, Transfection

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: Characterization of an AAV6.2FF-hACE2 transduction model for SARS-CoV-2 infection of wild-type mice (A) Diagram of AAV genome expressing hACE2 from the CASI promoter. (B) Western blot of HEK293 cells transduced with AAV6.2FF-hACE and probed with an anti-hACE2 antibody. (C) BALB/c mice were administered 1 x 10 11 vg of AAV-Luc intranasally and imaged 10 days later using an IVIS imager. (D–F) (D) IFA images of lungs harvested from BALB/c mice infected intranasally with 1 x 10 11 vg of AAV-hACE2 or AAV-Luc and euthanized 10 days later. Lungs were stained with a rabbit anit-hACE2 antibody and imaged at 20 X (scale bar, 50mM). Viral RNA (E), and virus TCID50 titers (F) were determined in respiratory tissues on days 2 and 4 post-infection. n = 6 (3M, 3F). Statistical significance determined by Mann-Whitney test. ∗ = p < 0.05, ∗∗ = p < 0.01.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Transduction, Infection, Expressing, Western Blot, Staining, MANN-WHITNEY

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: SARS-CoV-2 spike DNA antigens protect from viral replication in vivo (A) Mice were immunized once or twice separated by four weeks with 10ug of pS via electroporation. Serum was collected at day 18 post-final immunization. At 35 days post-final immunization mice were infected intranasally with adeno-associated virus expressing human ACE2 (white). 17 days following AAV6-ACE2 transduction, animals were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 VIDO-01 P2. Four days post infection, animals were sacrificed to quantify viral replication. SARS-CoV-2 specific serum IgG endpoint titers (B) and pseudoviral neutralization titers (C) at day 18 post-final immunization. Replication competent virus (D), and viral RNA (E) in the lungs four-days post-infection. Pearson correlations between virus titer and serum IgG endpoints (F) and neutralization titers (G). Pearson correlations between viral RNA copies and serum IgG endpoints (H) and neutralization titers (I). Each point represents the average of duplicate samples from an individual animal, bars represent the mean, lines represent the median, and error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test (A and B), or Kruskall-Wallis ANOVA (D and E). Spearman correlations were used to determine relationships (F-I). Data are representative of one experiment with n = 5 males (squares) and 5 females (circles) per group.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: In Vivo, Electroporation, Infection, Expressing, Transduction, Neutralization

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet:

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunospot, Software

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: ACE2 orthologs of diverse species mediate SARS-CoV-2 infection. (A) 293T cells transfected with plasmids expressing 10 ACE2 orthologs or an empty vector and then infected by icSARS-CoV-2-mNG at a multiplicity of infection of 1 for 48 h. Images were captured by using a Zeiss LSM 880 laser scanning microscope. (B) Quantification of infection by flow cytometry. Cells from panel A were fixed in 4% paraformaldehyde, and mNG-positive cells were then quantified by flow cytometric analysis. SSC, side scatter. (C) The expression of ACE2 ortholog plasmids in 293T cells was detected using a mouse anti-V5 tag monoclonal antibody targeting the C-terminal V5 tag (in red). Green, anti-β-actin antibody. (D) Representative confocal images of Caco-2 and Calu-3 cells infected with icSARS-CoV-2-mNG at a multiplicity of infection of 1 for 48 h.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Laser-Scanning Microscopy, Flow Cytometry

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: PRRA-led proteolytic cleavage of SARS-CoV-2 spike protein and effect on fusion. (A) Organization of SARS-CoV-2 spike protein and sequence alignment at the S1/S2 site with RaTG13 and pangolin GX S proteins. SP, signal peptide; NTD, N-terminal domain; FP, fusion peptide; TM, transmembrane; CT, C terminus. (B) 293T cells transfected with SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat RaTG13 spike (RaTG13 S), an insertion mutant containing PRRA (RaTG13 S+PRRA), and the pangolin GX spike protein (Pangolin GX S) (lanes 3 to 7) and SARS-CoV-2 S-infected Vero E6, Calu-3, and Caco-2 cells (lanes 8 to 10). A 5-min exposure of this part of the gel is included. Red, anti-S antibody; green, anti-β-actin antibody. (C) Cell-cell fusion mediated by CoV S proteins. 293T cells expressing Stop‐Luc, ACE2, and/or ACE2/TMPRSS2 (acceptor cells) were mixed at a 1:1 ratio with donor cells expressing Cre, CoV S, or both to initiate cell‐cell fusion. Data are presented as means ± standard errors of the means (SEM). (D) 293T cells or 293T-hACE2 cells were infected by pseudovirus bearing SARS-CoV-2 S. Bald viruses without any viral envelope or VSV-Gpp were included as negative and positive controls. (E) Entry of MLV pseudoviruses bearing SARS-CoV-2 S or SARS-CoV-2 S ΔPRRA in Vero cells. **, P < 0.001; ***, P < 0.0001. RLU, relative luciferase units.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Sequencing, Transfection, Mutagenesis, Infection, Expressing, Luciferase

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: The PRRA insert at the S1/S2 site distinctly modulates cell susceptibility to SARS-CoV-2 pseudovirions. (A to C) Entry of HIV pseudotyped with SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat RaTG13 S, RaTG13 S+PRRA, and pangolin GX S into 293T cells transiently expressing ACE2 (A), Calu-3 cells (B), and Caco-2 cells (C). (D) Western blot analysis of pseudovirions bearing SARS-CoV-2 S, SARS-CoV-2 S ΔPRRA, bat RaTG13 S, RaTG13 S+PRRA, and pangolin GX S. Red, anti-p24 antibody; green, antispike antibody. (E) Infection by pseudovirions harboring the indicated viral glycoproteins in 293T cells transfected with ACE2 alone or in combination with TMPRSS2. Data are presented as means ± SEM. *, P < 0.01.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: The novel S1/S2 site alters the dependence of RaTG13 pseudovirus on ACE2 orthologs. Shown are the infectivities of pseudovirions harboring the indicated viral spike proteins on 293T cells transiently transfected with Chinese hamster ACE2 and bovine ACE2 (A), Syrian hamster ACE2 and human ACE2 (B), ferret ACE2 and pig ACE2 (C), Agm ACE2 and mouse ACE2 (D), and horseshoe bat ACE2 and Malayan pangolin ACE2 (E). Data are presented as means ± SEM. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001. Numbers above the asterisks denote the fold changes. (F) Binding of pseudovirions carrying SARS-CoV-2 S, RaTG13 S, and RaTG13 S+PRRA to full-length recombinant human and mouse ACE2 was quantified by qPCR (see Materials and Methods). Binding relative to that of bald virus is plotted.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Transfection, Binding Assay, Recombinant, Virus

Journal: Journal of Virology

Article Title: The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

doi: 10.1128/JVI.01751-20

Figure Lengend Snippet: The novel S1/S2 site alters the dependence of RaTG13 pseudovirus on ACE2 orthologs in the presence of TMPRSS2. 293T cells expressing each ACE2 ortholog with TMPRSS2 were infected by pseudoviruses harboring SARS-CoV-2 S (A), SARS-CoV-2 S ΔPRRA (B), RaTG13 S (C), RaTG13 S+PRRA (D), pangolin S (E), and VSV-G (F). Luciferase activities in cell lysates were determined at 48 h postinfection. Data are presented as means ± SEM. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, P < 0.00001.

Article Snippet: Other ACE2 genes were synthesized (GenScript Biotech) and subcloned into the pcDNA3.1(+) vector between the BamHI and XhoI sites with a C-terminal V5 tag.

Techniques: Expressing, Infection, Luciferase

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: a. Schematic of pooled screen to identify SARS-CoV-2 regulators in Vero E6 cells. b. Scatter plot showing the gene-level mean z-scores of genes when knocked out in Vero E6 cells. The top genes conferring resistance to SARS-CoV-2 are annotated and shown in blue. c. Comparison between this Vero E6 screen to the Vero E6 screen conducted by the Wilen lab 27. Genes that scored among the top 20 resistance hits and sensitization hits in both screens are labeled. d. Venn diagram comparing hits across screens conducted in Vero E6, A549, and Huh7 (or derivatives) cells (ectopically expressing ACE2 and TMPRSS2 or not) 23–28. The top 20 genes from each cell line are included, with genes considered a hit in another cell line if the average z-score was > 3.

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Comparison, Labeling, Expressing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: Properties of SARS-CoV-2 host factor screens assayed by cell viability. For each library, the number of unique guides per gene is indicated in parentheses. The essential gene QC serves as a metric for screen quality (see Methods ) in the untreated arm, when applicable; the number of days post-library introduction until the end of the experiment is written after the semicolon.

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques:

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: Calu-3-Cas9 cells were stably transduced to express 2 different sgRNAs (g1, g2) per indicated gene and selected for 10–15 days. a. Cells were infected with SARS-CoV-2 bearing the mNG reporter and the infection efficiency was scored 48h later by flow cytometry. b. The expression levels of ACE2 were analyzed by immunoblot, Actin served as a loading control. c. Relative surface ACE2 expression was measured using a Spike-RBD-Fc fusion and a fluorescent secondary antibody followed by flow cytometry analysis. d. Cells were incubated with SARS-CoV-2 at MOI 5 for 2h at 37°C and then treated with Subtilisin A followed by RNA extraction and RdRp RT-qPCR analysis as a measure of viral internalization. e. Cells were infected with Spike del19 and VSV-G pseudotyped, GFP expressing VSV and infection efficiency was analyzed 24h later by flow cytometry. f. Cells were infected with SARS-CoV-2 at MOI 0.05 and, 24h later, lysed for RNA extraction and RdRp RT-qPCR analysis. g. Aliquots of the supernatants from F were harvested and plaque assays were performed to evaluate the production of infectious viruses in the different conditions. h. Cells were infected with MERS-CoV and 16h later, infectious particle production in the supernatant was measured by TCID 50 . The mean and SEM of at least 5 ( a ), 3 ( c , d , e , f , h ) independent experiments or representative experiments ( b and g ) are shown. The red dashed line represents 50% inhibition ( a , c-f ).

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Stable Transfection, Infection, Flow Cytometry, Expressing, Western Blot, Control, Incubation, RNA Extraction, Quantitative RT-PCR, Inhibition

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: sgRNA sequences used for CRISPR KO perturbations

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: CRISPR, Sequencing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: sgRNA sequences used for CRISPRa perturbations

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Sequencing

Journal: Zoological Research

Article Title: Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

doi: 10.24272/j.issn.2095-8137.2020.118

Figure Lengend Snippet: Characterization of in-vitro transfection of hACE2 and SARS-CoV-2 infection in MLE-12 cells

Article Snippet: At approximately 80% confluent, the cells were either transfected with the human ACE2 (hACE2) expression plasmid pCMV-ACE2-GFPSpark or the control pCMV3 plasmid using FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s instructions.

Techniques: In Vitro, Transfection, Infection

Journal: Zoological Research

Article Title: Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

doi: 10.24272/j.issn.2095-8137.2020.118

Figure Lengend Snippet: In vivo hACE2 pulmonary transfection in mice

Article Snippet: At approximately 80% confluent, the cells were either transfected with the human ACE2 (hACE2) expression plasmid pCMV-ACE2-GFPSpark or the control pCMV3 plasmid using FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s instructions.

Techniques: In Vivo, Transfection

Journal: Zoological Research

Article Title: Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

doi: 10.24272/j.issn.2095-8137.2020.118

Figure Lengend Snippet: Pulmonary SARS-CoV-2 infection in hACE2-transfected mice

Article Snippet: At approximately 80% confluent, the cells were either transfected with the human ACE2 (hACE2) expression plasmid pCMV-ACE2-GFPSpark or the control pCMV3 plasmid using FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s instructions.

Techniques: Infection, Transfection

Journal: Zoological Research

Article Title: Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

doi: 10.24272/j.issn.2095-8137.2020.118

Figure Lengend Snippet: Comparison of SARS-CoV-2 infection in hACE2-transfected WT mice and CXCL5 −/− mice

Article Snippet: At approximately 80% confluent, the cells were either transfected with the human ACE2 (hACE2) expression plasmid pCMV-ACE2-GFPSpark or the control pCMV3 plasmid using FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s instructions.

Techniques: Comparison, Infection, Transfection