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: BMC Genomics

Article Title: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

doi: 10.1186/s12864-022-08612-7

Figure Lengend Snippet: Cellular model and targets selection. A Transdifferentiation of BLaER1 pre-B cells into macrophages is accompanied by a dynamic transcriptomic remodeling of the cells. BLaER1 lymphocytes transdifferentiate into functional macrophages in the presence of Interleukin 3 (IL-3) and Macrophage colony-stimulating factor (M-CSF) upon β-estradiol induced release of CEBPaER to the nucleus. B Flow cytometry analysis of cell surface markers at T0, T3 (3 days) and T6 (6 days) after induced transdifferentiation in the BLaER1-Cas9 cell line. During the process, BLaER1 cells progressively lose the CD19 (B-cell marker staining -X-axis-) and gain the Mac1 (macrophage marker staining -Y-axis-). C Merged k-means clustered expression profiles (color code) of peaking and upregulated genes during transdifferentiation: 16 initial clusters of lncRNA ( n = 174) and 36 initial clusters of protein coding genes ( n = 939). FPKM values were log10 transformed before the normalization to z-score. Each line shows the expression pattern of a gene along transdifferentiation. The color corresponds to the k-means cluster to which the gene belongs (see also Supplementary Fig. S and S )

Article Snippet: The library is available at Addgene.org (BLaER1 pgRNA CRISPR library ID 183825).

Techniques: Selection, Functional Assay, Flow Cytometry, Marker, Staining, Expressing, Transformation Assay

Journal: BMC Genomics

Article Title: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

doi: 10.1186/s12864-022-08612-7

Figure Lengend Snippet: pgRNA CRISPR library for lncRNA and pc-genes. A (Upper panel) Diagram of the CD19 gene indicating the target sequence of CD19 pgRNAs (sgRNA1 and sgRNA2, from left to right). (Lower panel) Flow cytometry analysis of fluorescence intensity of the CD19 protein in BLaER1-Cas9 cells infected with sgRNAs and pgRNAs. The relative Stain Index of the different infected cells compared to the maximum expression level of CD19 in control cells (BLaER1-Cas9 cells infected with pDECKO-GFP ) is represented. CD19 expression is reduced between 30 and 95% upon infection of sgRNAs. The infection of pgRNAs induces a consistent reduction of CD19 signal up to 95% with all pgRNAs tested. B Schematic diagram showing the position of pgRNAs targeting lncRNAs (targeting the promoter and the transcription start site) and pc-genes (targeting coding exons). C CRISPR library composition (number of targets of each biotype and pgRNA pairs designed per target)

Article Snippet: The library is available at Addgene.org (BLaER1 pgRNA CRISPR library ID 183825).

Techniques: CRISPR, Sequencing, Flow Cytometry, Fluorescence, Infection, Staining, Expressing

Journal: BMC Genomics

Article Title: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

doi: 10.1186/s12864-022-08612-7

Figure Lengend Snippet: CRISPR-Cas9 screening in BLaER cells . A Workflow of the CRISPR screening experiment. The pDECKO plasmid library was transfected into HeK293T cells to obtain a library of lentivirus. BLaER1-Cas9 cells were infected at a low multiplicity of infection and double selected with antibiotics (Blasticidin and Puromycin) for 20 days. The infected cells were induced for transdifferentiation into macrophages for 3 days (T3) and 6 days (T6). Cells were labeled with antibodies against cell surface markers: CD19 (for B-lymphocytes) and Mac1 (for macrophages). Transdifferentiation status was assessed by flow cytometry. Transdifferentiated and delayed populations were isolated by Fluorescence-Activated Cell Sorting (FACS). B Flow cytometry analysis of BLaER1-Cas9 cells infected with the pDECKO_non-targeting control (left panels) and with the pDECKO_CRISPR-library (right panels) at T0, T3 and T6 of transdifferentiation. CD19 antibody, conjugated with BV510 fluorophore, was used to identify B-cells and Mac1 antibody, conjugated with PE-Cy7 fluorophore, was used to identify macrophages. Quadrants are as follows: Q1 (macrophage-like cells with presence of Mac1 and absence of CD19 surface markers); Q2 (transition cells with the presence of Mac1 and CD19); Q3 (background and not stained cells, negative for Mac1 and CD19); Q4 (lymphocyte B-like cells with the presence of CD19 and absence of Mac1 surface markers). The percentage of cells in each of the 4 quadrants is shown. The fraction of sorted cells showing a delay of transdifferentiation (“delayed” fraction) is marked in blue (gate P4), and sorted cells that differentiate at a normal pace (“differentiated” fraction) are marked in orange (gate P5). See also Supplementary Fig. S . C Workflow for processing the sorted cell populations for deep sequencing. Genomic DNA of sorted cells was extracted and PCR amplified in two steps. For the first PCR, specific staggered primers were used to amplify the integrated fragment which contains the pgRNAs. For the second PCR, Illumina barcoded primers were used to pool different samples (see also Supplementary Fig. S ). Samples were sequenced by 150 bp paired-end Illumina sequencing. DDE (differentiation delayed effect) was calculated as the ratio of pgRNA counts in the delayed population versus the counts in the transdifferentiated population

Article Snippet: The library is available at Addgene.org (BLaER1 pgRNA CRISPR library ID 183825).

Techniques: CRISPR, Plasmid Preparation, Transfection, Infection, Labeling, Flow Cytometry, Isolation, Fluorescence, FACS, Staining, Sequencing, Amplification

Journal: BMC Genomics

Article Title: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

doi: 10.1186/s12864-022-08612-7

Figure Lengend Snippet: FURIN and NFE2 expression after CRISPR edition. A FURIN RNA and protein expression. Cells were collected at T0 (before induction) and T3 (3 days after transdifferentiation induction). (CT0) and (CT3) negative control pDECKO-Intergenic at T0 and T3 respectively, (FUT0) and (FUT3) pDECKO-FURIN at T0 and T3, (FUT3s) pDECKO-FURIN at T3 and sorted from gate P4 (delayed population). Upper panel, qRT-PCR to check the expression of FURIN using two different sets of primers. Results are normalized to GAPDH and the fold change is calculated relative to the expression of cells infected with pDECKO-intergenic pgRNA at T3. The expression of FURIN decreases in cells infected with FURIN pgRNAs, especially in the delayed subpopulation (FUT3s). Bottom panel, western blot to assess the levels of the FURIN protein in BLaER1-Cas9 infected cells. Anti-FURIN antibodies recognize a band (marked with an arrowhead), the signal of which increases at T3, in line with RNA-Seq data (Supplementary Table S4). The FURIN band is not detectable in the pDECKO-FURIN infected cells (FUT3 and FUT3s). Uncropped blots are shown in Supplementary Fig. S A. B NFE2 RNA and protein expression. (CT0) and (CT2) negative control pDECKO-Intergenic at T0 (before induction) and T2 (2 days after transdifferentiation induction) respectively, (NFT0) and (NFT2) pDECKO-NFE2 at T0 and T2, (NFT2s) pDECKO-NFE2 at T2 and sorted from gate P4 (delayed population). Upper panel, qRT-PCR to check the expression of NFE2 using 2 different sets of primers. Results are normalized to GAPDH and the fold change is calculated relative to the expression of cells infected with pDECKO-intergenic T2. NFE2 expression in NFE2 pgRNA targeted cells is higher than in intergenic control cells (NFT2 and NFT2s compared to CT2). Bottom panel, western blot to check the protein levels of NFE2 in BLaER1-Cas9 infected cells. Anti-NFE2 antibodies detect two bands, the signal of which increases at T2 (CT2 compared to CT0). These two bands are strongly reduced in NFE2 targeted populations (NFT2 and NFT2s compared to CT2). Uncropped blots are shown in Supplementary Fig. S B

Article Snippet: The library is available at Addgene.org (BLaER1 pgRNA CRISPR library ID 183825).

Techniques: Expressing, CRISPR, Negative Control, Quantitative RT-PCR, Infection, Western Blot, RNA Sequencing Assay