custom single-guide rna (sgrna) lentiviral library  (Cellecta 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: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

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: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

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. 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: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

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

Journal: Frontiers in Immunology

Article Title: RBM39 shapes innate immunity by controlling the expression of key factors of the interferon response

doi: 10.3389/fimmu.2025.1568056

Figure Lengend Snippet: A genome-wide CRISPR/Cas9 screen identifies potential TLR3-related factors. (A) Schematic of the lentiviral truncated BID (tBID) death reporter system. The tBID expression is driven by the IFIT1 promoter, while the expression of the neomycin selection marker (NeoR) is under the control of the constitutive PGK promoter. Working principle: (1) dsRNA is taken up via endocytosis and transported to the endosome. (2) The endosomal TLR3 recognizes dsRNA and triggers signaling via TRIF, resulting in the activation of transcription factors IRF3 and NF-κB. (3) Activated transcription factors induce the expression of tBID by binding the IFIT1 promoter. (4) tBID associates with Bcl-2 proteins BAX and BAK to form a complex that permeabilizes the outer mitochondrial membrane and mediates the release of cytochrome c (Cyt c) into the cytoplasm. (5) Cyt c binds APAF1, triggering its oligomerization and binding to Pro-Caspase-9. Activated Caspase-9, in turn, activates Caspase-3 and -7, inducing apoptosis. (B) Workflow of the CRISPR/Cas9 screen. PH5CH and Huh7-Lunet-TLR3 stably expressing the death reporter and Cas9 were transduced with the genome-wide lentiviral CRISPR sgRNA library and selected with puromycin for stable expression. TLR3 stimulation with poly(I:C) induced the expression of tBID and thus apoptosis in case of intact TLR3 signaling. Then, surviving TLR3-deficient cells were enriched and collected for next-generation sequencing (NGS). (C) Illustration of the filtering steps to generate the final hit list. For each experiment independently, the ~2,000 most enriched gRNAs were determined in the NGS dataset and matched to their respective target genes, creating a list of 2,154 initial genes of interest. Subsequently, it was only focused on protein-coding genes. To ensure reproducibility, only genes with enriched gRNAs in three out of four repetitions were considered; this also eliminated genes showing enrichment in only one of the cell lines used. Lastly, genes were filtered to have at least five out of six available gRNAs enriched in at least one repetition to remove hits driven by potential off-target effects of just a subset of gRNAs. (D–F) A total of 50 candidate genes were selected and subjected to siRNA silencing for 48 h; then, the cells were stimulated with 50 μg/mL poly(I:C) supernatant feeding for 8 h. IFIT1 mRNA was measured by RT-qPCR normalized to GAPDH and expressed as relative expression to siRNA non-targeting (siNT)-treated samples. Knockdown of the candidates upregulated (D) or downregulated (E) IFIT1 mRNA expression in Huh7-Lunet-TLR3 cells. Knockdown of RBM39 , PCF11 , PTPRT , and KDM2A in PH5CH cells reduced IFIT1 mRNA expression in PH5CH cells (F) . siUNC93B1, siTLR3, and siTRIF were used as positive controls. The data are from three biological replicates ( n = 3); error bars indicate standard deviation (SD).

Article Snippet: After that, PH5CH and Huh7-Lunet-TLR3 cells stably expressing the tBID death reporter and Cas9 were transduced with lentiviral vectors containing the GeCKOv2.0 human-genome-wide sgRNA library ( ) (Addgene, USA) at MOI = 0.3.

Techniques: Genome Wide, CRISPR, Expressing, Selection, Marker, Control, Activation Assay, Binding Assay, Membrane, Stable Transfection, Transduction, Next-Generation Sequencing, Quantitative RT-PCR, Knockdown, Standard Deviation