human genome wide crispra sgrna library  (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: Ribosome-associated quality control of aberrant protein production during amino acid limitation

doi: 10.64898/2026.01.14.699605

Figure Lengend Snippet: (A) A dual reporter system with a Flag-mCherry CGG -4xCGG-DHFR CGG (internal control, no translation disruption) and a Flag-YFP CGG -4xAGA-DHFR CGG was integrated into the AAVS1 locus in cells expressing CRISPRi or CRISPRa machinery (see Methods) to query the effect of a genome-wide library of CRISPRi/a sgRNAs on specific translation disruption signal. Flow cytometry was used to sort high and low responding populations. (B-D) Total score (phenotype score * Mann-Whitney (M-W) p-value, see Methods) versus hit rank for highest (high YFP) and lowest (low YFP) scoring genes in K562 CRISPRi (B), 293T CRISPRa (C), and 293T CRISPRi (D) screens. Hits are labeled and colored by associated function. Dashed line marks score cut-off where FDR = 0.25. (E) Diagram connecting major screen hit pathways. Hits are colored by direction of phenotype; blue gene labels reduce translation disruption product accumulation, red gene labels increase translation disruption product accumulation.

Article Snippet: Genome-wide CRISPRi and CRISPRa sgRNA libraries (hCRISPRi_v2: Addgene #83969 and #83970; hCRISPRa_v2: #83978 and #83979) were amplified, packaged into lentiviral particles, and titers were determined as described in ref . For K562, CRISPRi parental cells (187 million) were infected at a multiplicity of infection (MOI) of 0.28 and selected using 2 μg/mL puromycin (Sigma) for 6 days starting 48 h post-transduction.

Techniques: Control, Disruption, Expressing, Genome Wide, Flow Cytometry, MANN-WHITNEY, Labeling

Journal: bioRxiv

Article Title: Ribosome-associated quality control of aberrant protein production during amino acid limitation

doi: 10.64898/2026.01.14.699605

Figure Lengend Snippet: (A) Schematic outlining how the screen with reporters and sorting scheme depicted in was performed. Cells were arginine limited for 3 days, sorted, recovered, and re-sorted into the same bin after a second period of arginine limitation. After recovery, guide RNAs were sequenced to calculate enrichment scores. (B,C) Western blot (B) and flow cytometry (C) validation of selected hits with negative or positive phenotype scores across various pathways in K562 cells. Cells expressing CRISPRi targeting hits (or non-targeting controls; NTC) and dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ) were arginine limited for 3 days to assess translation disruption product levels (B,C) and signaling responses through mTOR, GCN2 and ZAKα (B). In (B), “*” marks non-specific band from blot stripping and reprobing. (D) Western blot to assess translation disruption and GCN2 response in 293T cells overexpressing GADD34 or an NTC by CRISPRa (scFv-sfGFP-GCN4-VP64) and dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ), with or without limitation for arginine for 5 days and treatment with 40 nM ISRIB. (E) Western blot to assess translation disruption with or without limitation for leucine or arginine for 7 days and treatment with 250 nM Torin1 in MiaPaCa cells expressing the Flag-YFP CGG -2xAGA-DHFR CGG reporter. (F) Flow cytometry to assess translation disruption product accumulation upon limitation for arginine with or without GCN2 knockdown by CRISPRi and 250 nM Torin1 treatment in K562 cells expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ). (G-H) Western blots to assess phospho-p38 and -JNK response to arginine or leucine limitation and 0.1 μg/mL anisomycin treatment with or without treatment with 5 μM SB202190 (p38 inhibitor) in wild-type and GCN2 KO 293T cells. (I) Western blot to confirm ZAKα KO in wildtype and GCN2 KO 293T cells as indicated. (J-K) Western blots to assess phospho-p38 and -JNK response to 0.1 μg/mL anisomycin treatment or UV irradiation (J) or arginine or leucine limitation (K) in wild-type and GCN2 KO, ZAKα KO, and ZAKα+GCN2 double KO 293T cells as indicated. (L) Flow cytometry to assess reporter fluorescence upon limitation for arginine for 6 days with or without treatment with 5 μM SB202190 in wild-type and GCN2 KO 293T cells expressing the Flag-YFP CGG -4xAGA-DHFR CGG (“AGA”) or Flag-YFP CGG -4xCGG-DHFR CGG (“CGG”) reporter as indicated. (M) Flow cytometry to assess reporter fluorescence over time upon limitation for arginine with or without treatment with 5 μM SB202190 in K562 cells with (sgGCN2) or without (sgNTC) GCN2 CRISPRi knockdown expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ). (N) Change in translation disruption reporter (Flag-YFP CGG -4xAGA-DHFR CGG (“AGA”) or Flag-YFP CGG -4xAGA-DHFR CGG (“CGG”)) mRNA level upon arginine limitation for 3 days with or without treatment with 5 μM SB202190 in 293T cells. (O) Change in translation disruption reporter mRNA level (Flag-YFP CGG -4xAGA-DHFR CGG ) upon arginine limitation for 3 days with (sgLAMTOR2) or without (sgNTC) CRISPRi knockdown of LAMTOR2 in 293T cells expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ), relative to cells expressing control guide (sgNTC 1). (C,L,N,O) Error bars represent standard error of the mean of 3 replicates. (B,D,E, G-K) Full-length reporter product is indicated (α-GFP antibody used to detect YFP, α-vinc = α-vinculin).

Article Snippet: Genome-wide CRISPRi and CRISPRa sgRNA libraries (hCRISPRi_v2: Addgene #83969 and #83970; hCRISPRa_v2: #83978 and #83979) were amplified, packaged into lentiviral particles, and titers were determined as described in ref . For K562, CRISPRi parental cells (187 million) were infected at a multiplicity of infection (MOI) of 0.28 and selected using 2 μg/mL puromycin (Sigma) for 6 days starting 48 h post-transduction.

Techniques: Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Disruption, Stripping, Knockdown, Irradiation, Fluorescence, Control

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) Inhibition of anticancer immunity by Siglec receptors. Siglecs bind to sialic acid-containing glycans on the surface of target cells. B) Components of the Siglec-7 ligand structure. Disialyl-T glycans are organized in dense arrays on the backbone of specific mucin-type O-glycoproteins. C ) Targeted overexpression of genes by CRISPRa. A cell line expressing a dCas9-GCN4 peptide array and a VP64-SunTag fusion protein serves as the target cell line (K-562-CRISPRa cells). This cell line is lentivirally transduced with an sgRNA targeting the promoter region of a target gene. Subsequent recruitment of chromatin remodeling factors induces an increase in transcriptional activity. D) Recombinant Siglec-Fc proteins were precomplexed with an AlexaFluor647-antihuFc antibody (1 μg/mL) for 1 hour on ice. Precomplexes were subsequently incubated with K-562 cells for 30 minutes and analyzed by flow cytometry. Representative flow cytometry plots are depicted for each Siglec-Fc as well as a human Fc (hFc) negative control. E) K-562-CRISPRa cells were lentivirally transduced with a genome-wide library of ∼104,000 sgRNAs (5 sgRNAs/gene). After selection and propagation, 1.25 x 10 8 cells were then stained with Siglec-Fc reagents as in B . A high-binding (top 20%) and low-binding (bottom 20%) of the fluorescent population was then selected and sorted by FACS.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Inhibition, Over Expression, Expressing, Peptide Microarray, Transduction, Activity Assay, Recombinant, Incubation, Flow Cytometry, Negative Control, Genome Wide, Selection, Staining, Binding Assay

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) K-562-dCas9-CRISPRa cells were transduced with two different sgRNAs targeting the CD24 promoter region. After selection with puromycin, cells were stained with Siglec-10-Fc as in 1B and with a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. B) THP-1 cells were stained with Siglec-10-Fc as in 1B and a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. C) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting MGAT1 to generate a polyclonal cell population containing WT and MGAT1 KO cells. Cells were then co-stained with Siglec-10-Fc and the lectin L-PHA (5 μg/mL), which binds to complex N-linked glycans. Representative staining is indicated on the flow cytometry plot. D) Graph indicates MFI of Siglec-10-Fc staining in the WT (L-PHA+) and MGAT1 KO (L-PHA-) populations. E) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting C1GALT1. Cells were co-stained with Siglec-10-Fc and the lectin DBA, which binds to exposed α-GalNAc. Representative staining is indicated on the flow cytometry plot. F) Graph indicates MFI of Siglec-10-Fc staining in the WT (DBA-) and C1GALT1 KO (DBA+) populations. G) Pathway diagram indicates the enzymes involved in 2,3-linked and 2,6-linked sialylation of N-linked glycans. ST3GAL4, ST3GAL6 and ST6GAL1 were all strong hits in the Siglec-10 screen. H) The heat map indicates the average mRNA expression of key glycosyltransferase hits in cell lines derived from B-ALL, T-ALL, AML and multiple myeloma. mRNA expression values were extracted from DepMap and the Human Protein Atlas. I) OCI-AML-2-Cas9 cells were transduced an sgRNAs against ST3GAL4. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. J) The MFI of Siglec-10 staining in OCI-AML-2 WT and ST3GAL4 KO cells is indicated. MFI is internally normalized to the WT cell line. K) MM1S-Cas9 cells were transduced an sgRNAs against ST6GAL1. ST6GAL1 KO cells were isolated by FACS using the lectin SNA. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. L) The MFI of Siglec-10 staining in MM1S WT and ST6GAL1 KO cells is indicated. MFI is internally normalized to the WT cell line. M) K-562-CRISPRa cells were transduced and stained as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CD44 High” expression gate. N) The MFI of Siglec-10-Fc staining in NT cells vs. “CD44 High” cells is shown. MFI is internally normalized to the WT cell line. O) K-562 CRISPRa cells were transduced with an sgRNA against CSPG4 and co-stained with Sig10-Fc and a CSPG4 antibody as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CSPG4 High” expression gate. P) The MFI of Siglec-10 staining in NT cells vs. “CSPG4 High” cells is shown. MFI is internally normalized to the WT cell line. Statistical significance was determined using a two-tailed t-test. ** indicates p<0.01, * indicates p<0.05. Mean values plotted, error bars indicate SEM.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Transduction, Selection, Staining, Flow Cytometry, Expressing, Derivative Assay, Labeling, Isolation, Two Tailed Test

Journal: Oncogene

Article Title: In Vivo Genome-Wide CRISPR Screening Identifies CITED2 as a Driver of Prostate Cancer Bone Metastasis

doi: 10.1038/s41388-024-02995-5

Figure Lengend Snippet: Identification and analysis of metastasis hits in all genome-wide screens. (A) Schematic of samples sequenced to identify sgRNAs. (B) Scatter plots showing the representation of individual sgRNAs (shown as percentage of reads for each sgRNA over all reads in a sample) in metastasis (left) and their corresponding primary tumors (right). The dashed line shows the cutoff used for panels C-F. (C-F) Stacked bar graphs highlighting the identity of the “top-represented sgRNAs” (more than 5% of all reads in a particular metastatic sample) for each individual screen replicate. (G) Venn diagrams showing the overlap between significantly enriched sgRNAs in each metastasis compared to primary tumors, after Mageck-RRA analysis (FDR-corrected P<0.001). An arrow highlights CITED2 as the only overlapping sgRNA in CRISPRa screens. (H) Volcano plot of gene-level Mageck-RRA analysis in bone metastasis of the 22Rv1- Clone 1 - CRa screen highlighting CITED2 as the only significant hit (FDR-adjusted P=0.0025). Panel A was created with BioRender.com.

Article Snippet: Amplification of sgRNA libraries Genome-wide libraries (CRISPRa and CRISPRi v2 libraries with top5 sgRNAs/gene, Addgene #83978 and # 83969, respectively) were a kind gift from Dr Jonathan Weissman and were amplified by electroporation of 100ng DNA into Endura electrocompetent cells (Lucigen Corporation, Middleton, WI #60242) using an Amaxa Nucleofector II device (Lonza, Cologne, Germany) and Fisherbrand #FB101 electroporation cuvettes.

Techniques: Genome Wide