Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between pgRNA read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: CRISPR, Transduction, Selection, Control, Infection

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: Functional validation of prioritized oncogenic lncRNA candidates. (A) RP11-350G8.5 and LINC00467 basal expression levels via quantitative real time PCR (qRT-PCR) in MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors (values are normalized to the expression of GAPDH). (B) Representative image of genomic PCR products before and after KO of LINC00467 and RP11-350G8.5 in AMO-1 cells, visualized on 1.5% agarose gels. On the right: Sanger sequence of the amplicons encompassing the CRISPR-targeted region. Blue rectangles highlight pgRNA binding sites, whereas colored lines refer to the schematic picture of the KO reported above the gel picture (on the left). (C) Representative image of flow cytometric monitoring of AMO-1 and ABZB cells transduced with a SCRAMBLE-GFP-CRISPR vector (dark gray) or LINC00467/KO-GFP-CRISPR vector (light blue) or RP11-350G8.5/KO- GFP-CRISPR vector (red). Light-gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction) with overlapping colored curves at day 20. (D) Representative images of colony assay of AMO-1 and ABZB GFP-sorted cells, 15 days after plating, were generated using EVOS XL-Core microscope (Invitrogen by Thermo Fisher) (magnification ×10). (E) Number of colonies in 3 independent wells. (F) Dose-response curves 24 hours after treatment with bortezomib (1-10 nM). Percentage of viable cells ± standard deviation are normalized with respect to DMSO-treated cells (vehicle) for each experimental condition. Statistical differences were assessed across all plots via Student t test; ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing, CRISPR, Binding Assay, Transduction, Plasmid Preparation, Colony Assay, Generated, Microscopy, Standard Deviation

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: RP11-350G8.5 putative oncogenic role: in vitro validation and preliminary data from in vivo models. (A) Flow cytometric monitoring of GFP expression in ABZB cells transduced with a SCRAMBLE-GFP-CRISPR negative control vector, an RPL8 /KO-GFP-CRISPR positive control vector (selected from Project Score [37]), and 2 GFP-CRISPR constructs encoding for 2 pgRNAs targeting RP11-350G8.5. Gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction), while colored curves represent the percentage of viable cells at day 20. Bars on the right represent the fold change in percentage of GFP-expressing cells 20 days after target depletion against day 0. (B) Evaluation of IL-6R RNA expression level through quantitative real time PCR (qRT-PCR) on ABZB after transduction with SCRAMBLE vector or KO of RP11-350G8.5 with pgRNA#1 or pgRNA#2 or with a vector overexpressing RP11-350G8.5 (UP). (Data are normalized to the expression of GAPDH.) Statistics were obtained using multiple t -tests, resulting in no significant (ns) differences, as per the reported P values. (C) Flow cytometric monitoring of GFP in JJN.3 and NCI-H929 MM transduced cells, and percentage of GFP-positive cells is reported by overlapping curves referred to day 20 (colored curves) against day 0 (light gray curves). (D) Validation of RP11-350G8.5 KO in nontumoral cells, performed as described for A and C. (E) Representative images of RNA-FISH analysis. Nuclei are counterstained with DAPI (blue signal), whereas C3-fluorescein–conjugated GAPDH (green signal) has been used as cytoplasmic marker. Customly designed Stellaris probes targeting RP11-350G8.5 have been conjugated with 5-carboxytetramethylrhodamine (TAMRA) dye (red signal). Representative pictures acquired with a DMI6000-AF6000 Leica (Wetzlar, Germany) fluorescence microscope at magnification ×63 are reported, followed by specific regions of interest (ROIs), which are represented as enlarged images. (F) Dose-response curves 24 hours after treatment with bortezomib in AMO-1 cells overexpressing RP11-350G8.5 (1-10 nM). Statistics were analyzed using multiple t -tests (cutoff ∗ P < .05, ∗∗ P < .01). (G) In vivo imaging of engrafted ABZB cells. A total of 5 × 10 6 ABZB cells, which previously underwent highly efficient transduction (multiplicity of infection = 1) of RP11-350G8.5 KO-GFP or the SCRAMBLE vectors, were subcutaneously inoculated in mice (n = 2 per group). Images of tumors were acquired when the tumoral masses became palpable (identified as DAY 1), and at the end of the experiment (DAY 16, when tumors reached 2 cm in diameter). Both DAY 1 and DAY 16 were set up by considering SCRAMBLE mice, because SCRAMBLE cells have been faster to generate tumoral masses, due to their higher proliferative rate, and to grow up to 2 cm in diameter, with respect to KO cells. Tumors appear as yellow high-density signals on the right flank of the mice. Pictures were obtained with the IVIS (Perkin Elmer) system. (H) Tumor growth as mean measurement ± standard deviation (SD) across mice groups (n = 2). (I) Photographs of excised tumors were captured by a digital camera. (J) Weights of excised tumors, reported as mean ± SD across mice groups. Statistics were analyzed using multiple t -tests (cutoff: ∗ P < .05).

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: In Vitro, Biomarker Discovery, In Vivo, Expressing, Transduction, CRISPR, Negative Control, Plasmid Preparation, Positive Control, Construct, RNA Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Fluorescence, Microscopy, In Vivo Imaging, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Intermolecular 3′UTR-3′UTR interactions drive Wnt gene activation through heteromeric protein assembly

doi: 10.64898/2026.05.05.723075

Figure Lengend Snippet: A. Schematic of β-catenin interaction with E-cadherin at the plasma membrane. B. Immunoblot analysis of total E-cadherin and β-catenin protein abundance in iPSCs. N = 3 clonal lines each were used, and Tubulin serves as loading control. C. Representative flow plots showing surface E-cadherin in control (dark gray), CTNNB1 KO (dark purple) and CTNNB1 Udel (dark blue) clones at the iPSC stage. The unstrained samples are shown in lighter colors. D. Quantification of the experiment from (C). Surface E-cadherin abundance was measured by MFI. Data are shown as mean ± SD from N = 3 independent experiments of N = 3 clonal lines, each. Welch’s t -test. ****, P = 2.5 x 10 -7 . E. Quantification of nuclear β-catenin relative to total β-catenin at 24h of DE differentiation from immunoblot analysis, shown in . Data are shown as mean ± SD from N = 2 independent experiments. Welch’s t- test. ns, not significant. F. Representative immunofluorescence images showing β-catenin distribution at 4h of DE differentiation. DAPI was used to stain the nucleus. Scale bar, 10 μm. G. Quantification of nuclear β-catenin from the experiment shown in (F). For each experiment, 8–10 random fields (>80 cells per experiment) were analyzed. Each dot represents the mean intensity from one experiment. Data are shown as mean ± SD from N = 3 independent experiments. Welch’s t -test. ns, not significant. H. Sanger sequencing of edited alleles of zebrafish ctnnb1 KO embryos. The guide RNA is underlined, and the cut site is indicated by the vertical dashed line. I. Representative gel image of genotyping PCR to validate zebrafish ctnnb1 3′UTR deletions using deletion-flanking primers. J. Schematic of the zebrafish ctnnb1 3′UTR deletion region. The predicted post-editing sequence is shown. Red arrowheads indicate the junction site after scarless repair. In mosaic embryos, edited PCR products were excised and analyzed by Amplicon sequencing. Sequences were aligned and composition is shown. Sequences matching the prediction are underlined; insertions are highlighted in red. K. As in , but additional zebrafish images are shown.

Article Snippet: The guide RNA pairs with the highest editing efficiency were purchased from Synthego (Table S6).

Techniques: Clinical Proteomics, Membrane, Western Blot, Quantitative Proteomics, Control, Clone Assay, Immunofluorescence, Staining, Sequencing, Amplification

Journal: bioRxiv

Article Title: Intermolecular 3′UTR-3′UTR interactions drive Wnt gene activation through heteromeric protein assembly

doi: 10.64898/2026.05.05.723075

Figure Lengend Snippet: A. Schematic of β-catenin-mediated activation of the Wnt transcriptional program. B. LEF1 mRNA expression at the indicated time points, normalized to GAPDH . Shown is mean ± SD rom N = 3 independent experiments. Welch’s t -test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. C. As in (B), but AXIN2 mRNA expression is shown. D. Immunoblot showing total β-catenin in Ctrl and Udel cells at the indicated time points. N = 2 clonal lines were examined. Tubulin serves as loading control. E. Immunoblot showing active and total β-catenin in Ctrl and Udel cells at DE 24h. N = 3 clonal lines were examined. Tubulin serves as loading control. F. Immunoblot showing nuclear and cytoplasmic β-catenin at DE 24h loaded at 1:1 ratio. Tubulin serves as loading control for cytoplasmic fraction and H3 serves as loading control for nuclear fraction. Quantification, see . G. Scatter plot showing log2FC in Udel versus Ctrl (x-axis) and log2FC in KO versus Ctrl (y-axis) at DE 24h of Wnt-responsive genes . Genes with significant changes (log2|FC| > 0.58 and FDR < 0.05) in both Udel and KO are shown in dark blue ( N = 231), whereas genes with significant change in KO only are shown in light blue ( N = 1933). Dashed lines indicate a FC of 1.5. Selected genes are indicated. Pearson’s correlation coefficient is shown. H. Gene ontology analysis of genes colored in (G). Bonferroni-corrected P values are shown. I. Shown is mean log2FC of Wnt-responsive genes with significant gene expression changes between DE 24h and stem cell state in Ctrl clones, stratified by the magnitude of induction or repression. The number of genes in the eight bins are 15, 21, 87, 290, 215, 75, 35, and 57. T-test for independent samples; ****, P < 2 x 10 -9 . J. For the genes from (I), mean log2FC in Udel versus Ctrl cells at DE 24h is shown. T-test for independent samples; *, P = 0.046; ***, P = 0.008. K. Schematic of 3′UTR loss-of-function approach of the zebrafish ctnnb1 gene. A genomic region of 776 bp is deleted using CRISPR-Cas9 and a pair of guide RNAs in fertilized eggs. At the mRNA level, this deletion results in partial deletion of the zebrafish ctnnb1 3′UTR. Embryonic defects are scored 72h after fertilization. Top panel, conserved nt between the human CTNNB1 and the zebrafish ctnnb1 3′UTR. Each line denotes an identical nt. L. Representative images showing a normal zebrafish embryo, injected with a non-targeting guide RNA (Ctrl), mild and severe abnormalities observed after injection of a pair of guide RNAs that generate a ctnnb1 3′UTR deletion (Udel) and severe abnormalities after injection of a guide RNA targeting the ctnnb1 coding sequence to generate a gene KO. Scale bar, 500 μm. M. Phenotype classification at 72h post-injection from experiment shown in (L). The total number of fish examined in each category is given. Shown is the mean fraction ± SD of the obtained phenotypes from three clutches obtained in two independent experiments. T-test for independent samples was performed; mild phenotype, uninjected (uninj) vs Udel, **, P =0.008; Uninj vs KO, ns; severe phenotype, uninj vs Udel, *, P = 0.046; uninj vs KO, ****, P = 4 x 10 -6 . N. Immunoblot showing total β-catenin obtained from zebrafish embryos at 72h post-injection. Four embryos were pooled for each sample. Actin was used as loading control. The numbers indicate relative protein abundance normalized to Actin in each sample. O. mRNA expression of lef1 and axin2 in zebrafish embryos 72 h post-injection, normalized to eef1 . Shown is mean ± SD of N = 3 mRNA preparations that each contained four different embryos. Welch’s t -test; *, P < 0.05; **, P < 0.01.

Article Snippet: The guide RNA pairs with the highest editing efficiency were purchased from Synthego (Table S6).

Techniques: Activation Assay, Expressing, Western Blot, Control, Gene Expression, Clone Assay, CRISPR, Injection, Sequencing, Quantitative Proteomics