lrg vector  (Addgene inc)

Journal: Cancer Research Communications

Article Title: Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening

doi: 10.1158/2767-9764.CRC-24-0416

Figure Lengend Snippet: CRISPRi screen for overexpressed lncRNAs in melanoma pathogenesis and expression of selected hits. A, Graphical outline of the conducted CRISPRi screen in the 501mel cell line. MOI, multiplicity of infection. B, Principal component analysis plot of samples used for the CRISPRi screen. D8 and D12 refer to selected 501mel-dCas9-KRAB single-cell clone and D7, D14. and D21 for the days of screening time. Negative control refers to no Dox treatment, and the plasmid library for lentivirus production was added as additional control. PC, principal component. C, Dot plots show results of the CRISPRi lncRNA library screen at time points day 7, 14, and 21 after selection and mCherry + /GFP + cells sort as CPM of control (= day 0) vs. respective time points. Each dot represents a single sgRNA. Red dots indicate significantly depleted sgRNAs and log 2 fc > −1. D, List showing top 10 depleted lncRNA (excluding PVT1 as not validated after screen) hits at day 21 vs. control day 0 as the mean log 2 fc of the top three depleted sgRNAs of each respective lncRNA. E, CRISPRi hit validation of top 2 to 3 individual sgRNAs of indicated lncRNAs; top: GFP competition assay in 501mel-dCas9-KRAB from days 4 to 24 by flow cytometry in percent GFP-positive cells. sgROSA (black curve) serves as a negative control. Curves show average GFP signal loss of sgRNAs shown in bottom: bar graphs of fold change GFP expression of individual sgRNAs relative to control at day 24. For color codes, see top panel. F, Boxplot showing the log 2 CPM expression of selected lncRNA CRISPRi screening hits RP11-267N12.3, RP11-120D5.1, BDNF-AS, XLOC_030781, and GMDS-AS1 in melanocytes ( n = 4), brain metastasis ( n = 5), and LN STCs ( n = 4) detected by RNA-seq. Ctrl, control; Mets, metastasis.

Article Snippet: Using a Gibson Assembly master mix (New England Biolabs), we cloned sgRNAs into a lentiviral sgRNA GFP-tagged vector (LRG; Addgene, #65656).

Techniques: Expressing, Infection, Negative Control, Plasmid Preparation, Control, Selection, Biomarker Discovery, Competitive Binding Assay, Flow Cytometry, RNA Sequencing

Journal: Cancer Research Communications

Article Title: Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening

doi: 10.1158/2767-9764.CRC-24-0416

Figure Lengend Snippet: CRISPRi screen lncRNA hit functional characterization. A, Flow cytometry–based dotmplot showing GFP and mCherry signal as proxy for sgRNA and dCas9 expression (left). 7-AAD fluorescence–dependent cell cycle analysis in 501mel-dCas9-KRAB (top) and WM136a-dCas9-KRAB (bottom) cell lines upon sgBDNF-AS, sgXLOC_030781, and sgGMDS-AS1 CRISPRi knockdown (rigth). Note that the top panel is plotted linear and bottom logarithmic because of better cell cycle–gating parameters. B, Cell cycle phase quantification based on A in percent of cells ( n = 2). C, PARP cleavage apoptosis assay performed by Western blotting upon sgBDNF-AS, sgXLOC_030781, and sgGMDS-AS1 knockdown in 501meld-Cas9-KRAB and WM1361a-dCas9-KRAB Representative Western blots (top). GAPDH detection was used as a loading control; densitometric quantification of apoptosis as cleaved PARP/GAPDH protein ratio (bottom; average of n = 2). sgROSA served as negative control and etoposide apoptosis inducer as positive control. D, Re-analysis of 7-AAD cell cycle assay staining using exact same gating parameters for sub-G 1 cellular fraction indicative of apoptosis. E, Transwell cell migration assay. Fluorescence photomicrographs show calcein-stained migrated cells of sgBDNF-AS, sgGMDS-AS1, and sgXLOC_030781 knocked-down 501mel-dCas9-KRAB cells vs. sgROSA negative control and quantification of average sum count cell numbers ( n = 2).

Article Snippet: Using a Gibson Assembly master mix (New England Biolabs), we cloned sgRNAs into a lentiviral sgRNA GFP-tagged vector (LRG; Addgene, #65656).

Techniques: Functional Assay, Flow Cytometry, Expressing, Fluorescence, Cell Cycle Assay, Knockdown, Apoptosis Assay, Western Blot, Control, Negative Control, Positive Control, Staining, Cell Migration Assay