human crispr pooled genome-wide brunello sgrna library  (Addgene inc)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR screen reveals modifiers of rAAV production including known rAAV infection genes playing an unexpected role in vector production

doi: 10.1016/j.omtm.2025.101408

Figure Lengend Snippet: Intracellular staining for assembled AAV2 capsids enables FACS-based measurement of rAAV production (A) Schematic representation of the workflow. HEK 293 cells are transfected with a transfer plasmid, AAV2 Rep/Cap, and Ad-Helper. Following triple transfection, the cells are either lysed or fixed. Cell lysis allows for the purification and quantification of AAV2 particles. Fixation followed by staining for intact AAV2 particles allows for detection of AAV2 levels by flow cytometry. (Created in BioRender. ODriscoll, E. (2025) https://BioRender.com/f36g430 ). (B) Mock transfected, Rep/Cap-only transfected, and triple transfected cells were fixed and stained using a primary antibody specific to intact AAV2 capsids. Flow cytometry data validated that the antibody specifically recognized assembled capsids but not unassembled Rep/Cap. (C) Screening paradigm for the genome-wide FACS-based CRISPR KO screen to identify genetic regulators of rAAV productions. HEK 293 cells were transduced first with Cas9 and then with a genome-wide sgRNA library. Following selection and expansion, the cells were triple transfected. At 72 h post-transfection, the cells were fixed and stained for assembled AAV2. The cells were then sorted, and the least fluorescent and most fluorescent cells were collected for sequencing and subsequent analysis. (Created in BioRender. ODriscoll, E. (2025) https://BioRender.com/f36g430 ). (D) Volcano plot displaying the phenotype on the x axis and statistical significance on the y axis with the top depleted and enriched genes annotated. (E) STRING analysis showing the protein-protein interaction clustering of the top 100 depleted genes (MCL inflation parameter = 1.5; only clusters with PPI enrichment p < 0.001 shown). Cluster 1, indicated in light blue, had significantly more interactions than expected by chance. Significantly enriched Gene Ontology Biological Process terms for cluster 1 are provided and show a strong enrichment in genes implicated in a proteoglycan biosynthetic process (bsp). (F) STRING analysis showing the protein-protein interaction clustering of the top 100 enriched genes (MCL inflation parameter = 1.5; only clusters with PPI enrichment p < 0.001 shown). Cluster 1, indicated in red, had significantly more interactions than expected by chance. Significantly enriched GO cellular component terms for cluster 1 are provided and show an enrichment in genes involved in protein transport.

Article Snippet: Brunello genome-wide sgRNA library containing an average of 4 sgRNAs per gene and 1,000 non-targeting control sgRNAs was purchased from Addgene (73178).

Techniques: Staining, Transfection, Plasmid Preparation, Lysis, Purification, Flow Cytometry, Genome Wide, CRISPR, Selection, Sequencing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR screen reveals modifiers of rAAV production including known rAAV infection genes playing an unexpected role in vector production

doi: 10.1016/j.omtm.2025.101408

Figure Lengend Snippet: Genes implicated in AAV infection are involved in rAAV production (A) The depletion arm of the rAAV production screen’s volcano plot is colored based on genes implicated in AAV infection from Pillay et al. (2016). (B) AAVR KO clonal HEK 293 line confirmed by western blot. (C) AAV-GFP transduction in wildtype HEK 293 and AAVR KO clonal lines. (D) Secondary screen of the top hits from the genome-wide rAAV production screen. The volcano plot is colored by the previously published AAV infectivity screen. (E) FACS-based quantification of rAAV production in polyclonal KO lines generated using lentiviral-delivered Cas9 and sgRNA. Two sgRNAs were tested per gene (denoted with circle and square points) in duplicate for a total of four replicates per gene. A representative FACS plot from one replicate is shown in addition to the normalized median fluorescent intensities. B3GAT3 ( p = 0.0003), B4GALT7 ( p < 0.0001), and EXT1 ( p = 0.0383) all significantly decreased rAAV levels compared with CLYBL. (F) Polyclonal EXT1 KO lines were generated by electroporation of Cas9/sgRNA RNPs and compared with HEK 293 cells electroporated with only Cas9. Editing efficiency was quantified by Sanger sequencing followed by ICE analysis. (G) rAAV production was measured by FACS for three EXT1 KO lines and three control lines. A t test was performed, and there was a significant ( p = 0.0005) decrease in median fluorescent intensity upon EXT1 KO compared with control. (H) rAAV production was measured by qPCR for six EXT1 KO lines and six control lines. A t test was performed, and EXT1 KO led to a significant ( p = 0.0152) decrease in viral levels. (I) AAV particles containing GFP as the transgene were produced in three EXT1 polyclonal KO lines. This AAV-GFP was used to transduce wildtype HEK 293 cells, and transduction efficiency was assessed by flow cytometric-based quantification. Based on a t test, there was no significant difference in transduction efficiency for rAAV produced in EXT1 KO lines compared with control ( p = 0.2129). All error bars in this figure represent the standard deviation from the mean.

Article Snippet: Brunello genome-wide sgRNA library containing an average of 4 sgRNAs per gene and 1,000 non-targeting control sgRNAs was purchased from Addgene (73178).

Techniques: Infection, Western Blot, Transduction, Genome Wide, Generated, Electroporation, Sequencing, Control, Produced, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR screen reveals modifiers of rAAV production including known rAAV infection genes playing an unexpected role in vector production

doi: 10.1016/j.omtm.2025.101408

Figure Lengend Snippet: Network analysis identifies trafficking proteins that modulate AAV production (A) STRING analysis showing all potential protein-protein interactions (with interaction scores of >0.150) between the top 10 hits from the secondary screen where gene KO increased rAAV production. Significantly enriched Gene Ontology (GO) terms included many vesicle-related annotations. (B) FACS-based quantification of rAAV production in polyclonal KO lines generated using lentiviral-delivered Cas9 and sgRNA. Two sgRNAs were tested per gene (denoted with circle and square points) in duplicate for a total of four replicates per gene. A representative FACS plot from sgRNA 1 is shown in addition to the normalized median fluorescent intensities. (C) Polyclonal KO lines were generated by electroporation of Cas9/sgRNA RNPs and compared with HEK 293 cells electroporated with only Cas9. Editing efficiency was quantified by Sanger sequencing followed by ICE analysis. (D) rAAV production was measured by FACS for three KO lines each for TMED2, TMED10, and MON2 and compared with three control lines. (E) rAAV production was measured by qPCR for six KO lines per gene and compared with six control lines. Both TMED10 ( p = 0.0016) and MON2 ( p = 0.0001) significantly increased rAAV production. (F) Flow cytometric-based quantification of wildtype HEK 293 cells transduced with AAV-GFP produced in polyclonal KO lines. There was no significant difference in transduction efficiency for rAAV produced in TMED2 ( p = 0.9330), TMED10 ( p = 0.4918), or MON2 ( p = 0.7960) KO lines compared with control. All error bars in this figure represent the standard deviation from the mean.

Article Snippet: Brunello genome-wide sgRNA library containing an average of 4 sgRNAs per gene and 1,000 non-targeting control sgRNAs was purchased from Addgene (73178).

Techniques: Protein-Protein interactions, Generated, Electroporation, Sequencing, Control, Transduction, Produced, Standard Deviation

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A-D) Representative histograms (left) and bar plots (right) showing the surface levels of HLA-A2:AFP (A) or HLA-A2 (B) in human THP-1-Cas9-AFP-BFP cells, and the surface levels of H-2Kb:OVA (C) or H-2Kb (D) in mouse RN2-Cas9-OVA-BFP cells transduced with the indicated sgRNAs. (n=3) (E and F) Schematic of the T cell activation assay (E) and bar plot showing the IL-2 secreted by the B3Z T cell hybridoma incubated with sgRNA-transduced RN2-Cas9-OVA-BFP cells (F). (n=3) (G and H) Schematic of the mouse T cell killing assay (G) and bar plot showing the percentages of sgRNA-transduced RN2-Cas9-OVA-BFP cells killed by the OT-I T cells (H). (n=3) (I and J) Schematic of the human T cell killing assay (I) and bar plot showing the percentages of sgRNA-transduced NY-ESO-1-expressing THP-1-Cas9 cells killed by the NY-ESO-1 TCR-T cells (J). (n=3) (K) Schematic of the in vivo validations of SUSD6 functions in a mouse syngeneic AML model. (L-P) Quantification of the tumor volumes (L and O) and Kaplan-Meier survival curves (M and P) of immunocompetent (L and M) or CD8+ T cell-depleted (O and P) mice transplanted with sgRNA-transduced C1498-Cas9-GFP cells as described in (K). (for L and M: n=4 for sgNT and n=6 for sgSusd6; for O and P: n=5 for sgNT and n=6 for sgSusd6) (R) Violin plot of SUSD6 mRNA levels in normal HSPCs, MDS cells, and AML cells with different karyotypes from patient samples. HSPCs, hematopoietic stem and progenitor cells; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; MDS, myelodysplastic syndromes. Data were obtained from BloodSpot.51 (S) Survival of AML patients with high or low expression of SUSD6 in the TCGA-LAML cohort. (T) Pearson correlation of SUSD6 expression levels in AML cells and T cell activation signature in CD8+ T cells from the bone marrow immuno-microenvironments from AML patients. Data were generated by single-cell RNA-seq.53 Data are presented as the mean ± SEM. ns, not significant; *, p< 0.05; **, p< 0.01; and ***, p< 0.001 by two-tailed unpaired Student’s t-test (A-D, F, H, and J), two-way ANOVA for the last time point (L and O), or Log-rank Mantel-Cox test (M and P). Rosa26-targeting sgRNA (sgRosa, for human) and non-targeting sgRNA (sgNT, for mouse) were used as controls.

Article Snippet: Human pooled genome-wide sgRNA library (Brunello) sequences , Addgene , Addgene: 73179.

Techniques: Transduction, Activation Assay, Incubation, Expressing, In Vivo, Generated, RNA Sequencing, Two Tailed Test

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A) Representative western blots (left) and normalized band intensities (right) of HLA-A and B2m in sgRNA-transduced THP-1 cells. (n=5). (B) Schematic of the surface HLA-A2 internalization assay in sgRNA-transduced THP-1 cells. (C) Quantifications of the surface-remaining HLA-A2. (n=3) (D-F) Time course studies of the surface HLA-A2 expression on shRNA-transduced THP-1 cells treated with Cycloheximide (CHX, D), Bafilomycin A1 (BafA1, E), or Epoxomicin (Epox, F) (n=4). (G-J) Representative confocal images (top) and quantifications (bottom) of the surface-derived MHC-I colocalized with the plasma membrane markers (G and I) or the lysosomal marker (H and J) in THP-1 cells (G and H) or MutuDC cells (I and J) transduced with indicated shRNAs. At least 100 cells were quantified in each group. All scale bars: 5 μm. (K and L) Flow cytometric analyses of intracellular MHC-I storage (K) and recycle (L) in shRNA-transduced THP-1 cells as described in Figures S5H and S5I, respectively. (for K: n=6; for L: n=4) Data are presented as the mean ± SEM (A-F, K, and L) or box and whiskers with all data points (G-J). ns, not significant; ***, p<0.001 by two-tailed unpaired Student’s t-test (A), two-way ANOVA for the last time point (C-F, K, and L), or Mann-Whitney test (G-J). sgRosa and shRen were used as controls.

Article Snippet: Human pooled genome-wide sgRNA library (Brunello) sequences , Addgene , Addgene: 73179.

Techniques: Western Blot, Expressing, shRNA, Derivative Assay, Clinical Proteomics, Membrane, Marker, Transduction, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human pooled genome-wide sgRNA library (Brunello) sequences , Addgene , Addgene: 73179.

Techniques: Control, Purification, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Transfection, Immunoprecipitation, Activation Assay, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Double Knockout, shRNA, Real-time Polymerase Chain Reaction, Genome Wide, Plasmid Preparation, Software, Flow Cytometry