hcrispra v2  (Addgene inc)

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: (A) Summary of gene deletion vs. gene overexpression approaches. (B) Rationale for the present study. (C) Experimental outline of the present study. (D) HEK293 and Jurkat cells were inoculated with different volumes of Ebola or rabies pseudovirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This revealed that Jurkat cells are largely refractory to Ebola or rabies pseudovirus entry, relative to HEK293 cells. (E) Construction of a clonal Jurkat cell line, known as “Jurkat C6”, stably expressing a degron-tagged CRISPRa construct ( left ). Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence or absence of TMP (1 μM) for 3 days, followed by flow cytometry to detect hCD19 expression ( right ). (F) At each of the indicated points of the genome-wide CRISPRa screen, the cell population was challenged with Ebola or rabies pseudovirus, and cell infectivity was evaluated by flow cytometry. Upon successive rounds of the screen, the cell population became progressively more susceptible to infection to either Ebola or rabies pseudovirus, respectively. (G) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for Ebola pseudovirus entry. (H) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for rabies pseudovirus entry.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Over Expression, Marker, Mutagenesis, Flow Cytometry, Infection, Stable Transfection, Expressing, Construct, Transduction, Genome Wide

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) Plasmids used to pseudotype non-replicating lentiviruses with either Ebola or rabies envelope proteins. EBOV-GP: glycoprotein of Ebola virus, Makona variant. RABV-GP N2C: glycoprotein of rabies virus, N2C variant. B) HEK293 and Jurkat cells were inoculated with different volumes of VSV envelope protein-pseudotyped lentivirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This served as a positive control to confirm that Jurkat cells and HEK293 cells are both susceptible to VSV pseudovirus entry. C) Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence of different TMP concentrations (0-4 μM) for 2-3 days, followed by flow cytometry to detect human CD19 expression.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Virus, Variant Assay, Marker, Mutagenesis, Flow Cytometry, Infection, Positive Control, Transduction, Expressing

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. B) L-SIGN or DC-SIGN were expressed in Jurkat or primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of primary T cells to authentic Ebola and Sudan virus infection.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Expressing, Control, Flow Cytometry, Infection, Mutagenesis, Virus

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. As positive controls, flow cytometry was used to confirm successful delivery of the sgRNA construct as part of the CRISPRa workflow (as denoted by BFP expression) and that NGFR was expressed (upon cDNA expression). B) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. Cells expressing the highest levels of L-SIGN and DC-SIGN were preferentially infected by Ebola pseudovirus. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. On days 0, 1, and 2 post-infection, flow cytometry was performed to determine the percentage of infected cells and qPCR was performed on cell culture supernatants to quantify viral genome replication. This revealed that L-SIGN or DC-SIGN expression enabled authentic Ebola and Sudan virus entry into primary human T cells, but viral genome replication was impaired, perhaps reflective of cell-intrinsic restriction factors.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Expressing, Control, Flow Cytometry, Infection, Construct, Mutagenesis, Virus, Cell Culture

Journal: bioRxiv

Article Title: Antibody-Dependent Heterotypic Syncytia Drive COVID-19 Inflammation and Disease Progression

doi: 10.64898/2026.02.11.705426

Figure Lengend Snippet: A , Schematic of the CRISPR-Cas9 screen. B , Volcano plot of genes from the screen, ranked by fold change and adjusted P value. C , Representative flow cytometry dot plots of GW01-induced syncytia between A549-spike and FCER1G -KO (FcRγ-KO) THP-1 clones versus non-targeting control (NT), and quantification of syncytia(right inset)(mean ± SEM, n=3, * P <0.05, **** P <0.0001). D , Representative plots A549-spike fusion with ADAM10-KO THP-1 clones versus control and quantification of syncytia. E , Rescue of FcRγ: representative plots for FcRγ-KO-LVX (empty lentiviral vector), FcRγ-KO-FcRγ (FcRγ cDNA), and NT-LVX; with quantification of syncythia. F , Rescue of ADAM10: representative plots for ADAM10-KO-LVX (empty vector), ADAM10-KO-ADAM10 (ADAM10 cDNA), and NT-LVX, along with quantification of syncytia. G , Effect of CD64 or CD32 blocking antibodies on GW01-dependent syncytium formation measured by NanoLuc assay. H , Effect of the ADAM10 inhibitor GI254023X on GW01-dependent syncytium formation. I , Representative fluorescence images of syncytia (yellow merge) between THP-1-mCherry (red) and A549-spike (green; CFSE) at 3 hour post co-culture, with or without GI254023X. Scale bar, 100 μm. Data are presented as mean ± SEM, n=3, * P <0.05, *** P <0.001, **** P <0.0001 by one-way ANOVA.

Article Snippet: The genome-wide CRISPR sgRNA library (Addgene, 101926-101934) was packaged into lentiviruses.

Techniques: CRISPR, Flow Cytometry, Clone Assay, Control, Plasmid Preparation, Blocking Assay, Fluorescence, Co-Culture Assay