Journal: Journal of medical virology

Article Title: Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

doi: 10.1002/jmv.29087

Figure Lengend Snippet: FIGURE 1 Whole genome screening strategy for entry of SARS‐CoV‐2‐S, VSVG‐mNG, and VSVG‐CRE pseudoviruses. (A) ACE2 293T or ACE2‐293T‐GFPflox cells were transduced with the Brunello sgRNA genome‐wide CRISPR library. These cells were selected with puromycin to kill off cells that did not integrate the sgRNA cassette. Five days following selection, these cells were infected with SARS‐CoV‐2 Spike, VSV‐G, or VSV‐G‐Cre pseudoviruses and allowed 48 h for reporter expression. The cells were sorted using fluorescent activated cell sorting (FACS), genomic DNA was extracted, sgRNA sequences were amplified by PCR, sequenced by next‐generation sequencing, and sgRNA abundance in the sample was quantified by MAGeCK. (B) Representation of Cre‐LoxP system in ACE2‐293T‐GFPflox cells. The cells express dsRed and after excision of dsRed‐stop by Cre recombinase, the cells start to express EGFP.

Article Snippet: A CRE reporter was inserted into ACE2 293T cells using pLenti‐ V6.3 lox‐dsRED‐ stop‐lox‐eGFP‐blas (a gift from Ewa Snaar‐Jagalska, Addgene plasmid #106171).16 After selection with the Blasticidin, these cells were then FACS sorted based on their dsRED intensity.

Techniques: Transduction, Genome Wide, CRISPR, Selection, Infection, Expressing, FACS, Amplification, Next-Generation Sequencing

Journal: Journal of medical virology

Article Title: Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

doi: 10.1002/jmv.29087

Figure Lengend Snippet: FIGURE 2 Generation of ACE2 293T and CRE reporter ACE2 293T cell lines and their characterization. (A) 293T cells were transduced with lentivirus containing ACE2 cDNA for stable ACE2 293T cell line production. Cells were sorted after 72 h of transduction after staining for ACE2 using AF488 conjugated anti‐ACE2 antibody. The histogram shows ACE2 expression in these cells after sorting, red is the parental 293T cells and cyan is the ACE2 expressing 293T cells. (B) ACE2 293T cells were transduced with lentivirus containing CRE reporter gene and cells were selected with blasticidin to get the uniform population of dsRED expression. (C) GFP expression quantified after infection of ACE2 293T with Spike‐mNG and VSVG‐mNG or ACE2 293T‐GFPfloxed with VSVG‐CRE pseudoviruses. Spike‐mNG pseudovirus was concentrated to allow for titers similar to VSVG‐mNG.

Article Snippet: A CRE reporter was inserted into ACE2 293T cells using pLenti‐ V6.3 lox‐dsRED‐ stop‐lox‐eGFP‐blas (a gift from Ewa Snaar‐Jagalska, Addgene plasmid #106171).16 After selection with the Blasticidin, these cells were then FACS sorted based on their dsRED intensity.

Techniques: Transduction, Staining, Expressing, Infection

Journal: Journal of medical virology

Article Title: Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

doi: 10.1002/jmv.29087

Figure Lengend Snippet: FIGURE 4 Validation of key genes identified in CRISPR‐Cas9 whole‐genome screening using the one vector system. (A) ACE2 staining on the ACE2 KO cells using AF488 conjugated anti‐ACE2 antibody (dark blue) which is similar to unstained (magenta) versus WT ACE2 positive cells (light blue). (B) ACE2 staining on the ACE2‐293T‐CRE reporter cells following transduction with the ACE2 targeting sgRNA (dark blue) relative to WT (light blue) and unstained (magenta). (C–F) Effect of mutants on the entry of mNG and Cre reporter viruses. The error bar represents three biological replicates. One‐way ANOVA and Šídák's multiple comparisons test were performed to do statistical analysis between the WT and the mutants. Bar shows mean ± SD (n = 3). *Denotes p < 0.05 for comparison between WT and the mutants.

Article Snippet: A CRE reporter was inserted into ACE2 293T cells using pLenti‐ V6.3 lox‐dsRED‐ stop‐lox‐eGFP‐blas (a gift from Ewa Snaar‐Jagalska, Addgene plasmid #106171).16 After selection with the Blasticidin, these cells were then FACS sorted based on their dsRED intensity.

Techniques: Biomarker Discovery, CRISPR, Plasmid Preparation, Staining, Transduction, Comparison

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: 3P SARS2 VLPs are superior to 4P SARS2 VLPs. ( A ) 4P and 3P SARS2 Luc-PS9 VLPs were produced by co-transfection of either four or three plasmids into 293T producer cells. These plasmids encode for the viral structural proteins, along with a luciferase reporter coupled to PS9 packaging signal (Luc-PS9). ( B ) Luciferase assay showed ∼7-fold higher luminescence intensity in 293T-hACE2 cells upon using 3P versus 4P VLPs. ( C ) VLP concentrate (10 μl for M and E, 2 μl for S2 and N) was loaded in each lane. Western blot analysis suggests incorporation of all SARS2 structural components in VLPs, with 3P SARS2 Luc-PS9 VLPs displaying more intense protein bands compared with 4P VLPs. ( D ) Higher Luc-PS9 transcript levels were observed using RT-PCR in the case of 3P SARS2 Luc-PS9 VLPs. ( E ) Cryo-TEM images of 3P SARS2 Luc-PS9 VLPs showed spherical ∼100-nm-sized VLPs with double-layered membrane structures. ( F ) 3P SARS2 EGFP-PS9 VLPs produced with EGFP reporter efficiently infected 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Fluorescence images were acquired 24 h post-infection. ( G ) Flow cytometry VLP entry time-course showed peak fluorescence for 3P SARS2 EGFP-PS9 VLPs at 24 h followed by decrease at larger times. This was observed both for 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Abbreviations: aa, amino acid; nt, nucleotide. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: The pcDNA3.1 GFP-PS9-PS9 plasmid was cloned by inserting a second PS9 packaging sequence to the end of PS9 in pcDNA3.1 GFP-PS9. sgRNA targeting EGFP , SLC35A1 , and human ACE2 ( hACE2 ) were cloned into the original pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector provided by Kosuke Yusa ( RRID: Addgene_50946).

Techniques: Produced, Cotransfection, Luciferase, Western Blot, Reverse Transcription Polymerase Chain Reaction, Membrane, Infection, Fluorescence, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Tuning VLP viral tropism by altering viral glycoprotein. ( A ) Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike, or MERS spike) and reporter genes (luciferase or EGFP). ( B ) One microgram of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: WT 293T (293T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3, and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only hACE2-expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, and Calu-3), with SARS exhibiting higher luminescence intensity compared with SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types. ( C ) 3P EGFP-PS9 VLPs were produced with 4 μg VSV-G, 1 μg SARS2 spike, or 1 μg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provide detailed steps for VLP production. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: The pcDNA3.1 GFP-PS9-PS9 plasmid was cloned by inserting a second PS9 packaging sequence to the end of PS9 in pcDNA3.1 GFP-PS9. sgRNA targeting EGFP , SLC35A1 , and human ACE2 ( hACE2 ) were cloned into the original pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector provided by Kosuke Yusa ( RRID: Addgene_50946).

Techniques: Produced, Luciferase, Expressing, Infection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Streamlining VLP technology using a 2P system. ( A ) Four constructs were developed with two independent promoters driving expression of reporter gene and SARS2 structural proteins. The promoters were separated by insulator and terminator sequences to minimize promoter interference: synthetic polyA (spa); a G-rich sequence from β-actin (Tactb); chicken hypersensitive site 4 (cHS4); and a synthetic MAR sequence 8 (sMAR8) at the end of the E protein. (B, C) Each of these constructs was transfected into 293T cells along with spike plasmid to produce four different 2P SARS2 Luc-PS9 VLPs. 2P.2 VLPs displayed highest luminescence intensity ( B ). Its signal was comparable to 4P VLP but lower than 3P VLP ( C ). ( D ) Western blots of SARS2 structural proteins showed different patterns of protein expression for different 2P plasmids. 2P.2 SARS2 Luc-PS9 VLPs displayed more intense protein bands compared with 2P.3 and 2P.4, but this was lower than 2P.1. ( E ) 2P.2.EGFP VLPs were created by replacing the luciferase reporter with EGFP. VLP entry of 2P.2.EGFP SARS2 VLPs into A549-hACE2-TMPRSS2 and 293T-hACE2 cells was measured using flow cytometry. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: The pcDNA3.1 GFP-PS9-PS9 plasmid was cloned by inserting a second PS9 packaging sequence to the end of PS9 in pcDNA3.1 GFP-PS9. sgRNA targeting EGFP , SLC35A1 , and human ACE2 ( hACE2 ) were cloned into the original pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector provided by Kosuke Yusa ( RRID: Addgene_50946).

Techniques: Construct, Expressing, Sequencing, Transfection, Plasmid Preparation, Western Blot, Luciferase, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: VLPs deliver functional Cas9 mRNA into target cells to achieve gene editing. ( A ) 3P Cas9-P2A-dTo-T20 VLPs (dTo: dTomato) were used for gene editing studies, with surface glycoprotein encoding for either VSV-G or SARS2 spike. ( B ) General workflow of gene editing study performed in panels (C)–(E). sgRNAs were transfected into cells on day −1 using plasmids carrying BFP reporter. VLP-carrying spCas9 mRNA was introduced into cells on day 0. Tropism of the VLP depends on surface glycoprotein. Gene editing efficiency was quantified on day 6. Editing efficiency quantified percentage of BFP(+) population that either turned EGFP(−) (panels C and E) or lost ACE2 expression based on anti-hACE2 binding (panel D). ( C ) sgRNAs targeting EGFP were introduced into 293T-hACE2-EGFP and spCas9 mRNA was delivered using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( D ) sgRNAs against hACE2 were introduced to knockout the receptor in 293T-hACE2 cells using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( E ) sgRNAs targeting EGFP were introduced in 293T-EGFP cells, with genome editing being performed using 3P Cas9-P2A-dTo-T20 VLPs bearing either VSV-G or SARS2 spike. In all panels, the target gene ( EGFP or hACE2 ) was knocked out in 20%–35% of cells expressing sgRNA. Higher VLP amount resulted in greater editing. ( F ) 293T-hACE2 stably expressed sgRNAs against SLC35A1 were infected with 3P SARS2 Cas9-P2A-dTo-T20 VLPs (1.885 μg/μl N protein equivalent) or without SARS2 spike (1.385 μg/μl N protein equivalent). Gene editing efficiency was evaluated based on increase in fluorescent peanut agglutinin lectin (PNA) binding to cells. More than 70% gene editing was observed upon using 3P VLPs to edit endogenous genes. Volume of VLP used in each assay is specified in individual panels. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: The pcDNA3.1 GFP-PS9-PS9 plasmid was cloned by inserting a second PS9 packaging sequence to the end of PS9 in pcDNA3.1 GFP-PS9. sgRNA targeting EGFP , SLC35A1 , and human ACE2 ( hACE2 ) were cloned into the original pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector provided by Kosuke Yusa ( RRID: Addgene_50946).

Techniques: Functional Assay, Transfection, Expressing, Binding Assay, Knock-Out, Stable Transfection, Infection

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: In vivo pulmonary gene delivery using VLPs. ( A ) VLPs bearing either VSV-G or maSARS2 were instilled into mice at time = 0. Twenty-four hours post-instillation, luciferase activity was measured in tissue extracts from left lung, right lung, or trachea. Protein concentration in lysate was used to normalize luminescence signal, with untreated/mock values being set to 1.0 for all in vivo studies. ( B ) VSV-G VLPs were instilled via either o.p.a. (100 μl) or i.n. (50 μl) routes. o.p.a. resulted in VLP administration to mouse lung. ( C ) Q493K and N501Y mutations were introduced into SARS2 spike to generate maSARS2 spike. 3P VLPs with VSV-G, SARS2, maSARS2, and no spike were produced with N protein equivalents of 1.859, 1.149, 1.334, and 2.024 μg/μl, respectively. A total of 50 μl of VLP was used to infect three target cell types: 293T, 293T-hACE2, and 293T-mACE2. VSV-G VLPs infected all three cell types, SARS2 spike only infected 293T-hACE2 (hACE2), whereas maSARS2 spike was permissive to both 293T-hACE2 and 293T-mACE2. VSV-G luminescence was set to 1.0 in this panel. ( D ) Hundred microliters of VSV-G VLPs or maSARS2 VLPs, both 0.820 μg/μl N protein equivalent, were instilled via o.p.a. into mice. Whole lung tissue was harvested. Mice without VLPs served as negative control. Both VSV-G and maSARS2 VLPs enabled luciferase signal in mouse lung with VSV-G being more efficient. Data are mean ± STD. N = 5–6 for each mouse treatment group. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: The pcDNA3.1 GFP-PS9-PS9 plasmid was cloned by inserting a second PS9 packaging sequence to the end of PS9 in pcDNA3.1 GFP-PS9. sgRNA targeting EGFP , SLC35A1 , and human ACE2 ( hACE2 ) were cloned into the original pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector provided by Kosuke Yusa ( RRID: Addgene_50946).

Techniques: In Vivo, Luciferase, Activity Assay, Protein Concentration, Produced, Infection, Negative Control

Journal: Non-Coding RNA

Article Title: CRISPR/CasRx-Mediated RNA Knockdown Reveals That ACE2 Is Involved in the Regulation of Oligodendroglial Cell Morphological Differentiation

doi: 10.3390/ncrna8030042

Figure Lengend Snippet: Key antibodies and chemical sources. All key materials such as antibodies are shown.

Article Snippet: Cells were also transfected with the respective plasmids (pEF1a (pEF-BOS modified type) encoding CasRx and pSINmU6 encoding gRNA or pcDNA3.1-N-EGFP-human ACE2 intracellular domain) using the ScreenFect A transfection kit (FUJIFILM, Tokyo, Japan), according to the manufacturer’s instructions.

Techniques: Concentration Assay, Activation Assay, Fluorescence, Immunoprecipitation, Binding Assay, Transfection, Western Blot, Stripping Membranes, Recombinant, Expressing, Plasmid Preparation, Control

Journal: Non-Coding RNA

Article Title: CRISPR/CasRx-Mediated RNA Knockdown Reveals That ACE2 Is Involved in the Regulation of Oligodendroglial Cell Morphological Differentiation

doi: 10.3390/ncrna8030042

Figure Lengend Snippet: Knockdown of ACE2 by gRNA promotes oligodendroglial cell morphological differentiation. ( A , B ) FBD-102b cells were transfected with the plasmids encoding ACE2 gRNA with CasRx or control plasmids and were allowed to be differentiated for 0 or 3 days. Differentiation efficiencies were divided into three categories and depicted in graphs (**, p < 0.01; n = 5 fields). The number of branches in cells is also counted and shown (**, p < 0.01; 50 cells). ( C , D ) Cells at 3 days following the induction of differentiation were collected, lysed, and immunoblotted with an antibody against PLP1, CNPase, SOX10, or actin. Band intensities were also compared to be depicted in graphs (**, p < 0.01 and *, p < 0.05; n = 3 blots).

Article Snippet: Cells were also transfected with the respective plasmids (pEF1a (pEF-BOS modified type) encoding CasRx and pSINmU6 encoding gRNA or pcDNA3.1-N-EGFP-human ACE2 intracellular domain) using the ScreenFect A transfection kit (FUJIFILM, Tokyo, Japan), according to the manufacturer’s instructions.

Techniques: Knockdown, Transfection, Control