Figure 2 from Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening
Year: 2025
Type: other
Abstract: 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 log2 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 log2 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 log2 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.
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4412112333