lenti crispr v2 vector  (Addgene inc)

Journal: bioRxiv

Article Title: Osteocytes secrete adiponectin and display adipocyte-like phenotype under control of PPARG nuclear receptor

doi: 10.64898/2026.05.02.722443

Figure Lengend Snippet: Osteocytes accumulate and use lipids and express adipocyte-specific gene markers under control of PPARG. A. Visualization of lipid droplets in MLO-Y4 cells mock-or PPARG-edited with CRISPR/Cas9 (Y4 C and Y4 KO , respectively). Imaging using confocal microscopy, magnification 63×, lipids stained with BODIPY TM 493/503, as described in Material and Methods. Scale bars indicate 50 µm. B. Fuel preference (dependency and capacity) of MLO-Y4 cells measured with Seahorse FuelFlex assay as described in Material and Methods. Gluc. – glucose, FA – fatty acids, Glut. – glutamine. C. Relative expression of Perilipin family transcripts in osteocytes isolated from 3 mo old C57BL/6 male mice. Levels of expression Plin 2-5 were normalized to Plin1 . D. Relative levels of adipocyte-specific gene markers expression in MLO-Y4 osteocytes (Y4), OB6 and MC3T3 (MC) osteoblasts, and AD2 marrow adipocytes to which all values were normalized. Statistical significance was analyzed with One-way Anova with multiple comparison. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND – not detected.

Article Snippet: MLO-Y4 cells edited with CRISPR/Cas9 for PPARγ deletion were developed using editing system designed by Synthego CRISPR Gene Knockout Kit v2 (Synthego Corporation, Redwood City, CA; Cat # SO6765399) and selected single cell-derived clones were verified for the editing efficiency using ICE (Interference of CRISPR Edits), an online bioinformatic software provided by Synthego.

Techniques: Control, CRISPR, Imaging, Confocal Microscopy, Staining, Expressing, Isolation, Comparison

Journal: Cell Discovery

Article Title: Loss-of-function variants in ODAD1 disrupt ODA docking and induce actin cytoskeletal remodeling in primary ciliary dyskinesia

doi: 10.1038/s41421-026-00875-8

Figure Lengend Snippet: Video S20. Apical-out organoids-c.705_706insGCAG+Lentivirus

Article Snippet: After the primers were annealed, the sgRNA was ligated into the lenti CRISPR V2 vector (Addgene, cat# 52961) using T4 DNA ligase.

Techniques:

Journal: Cell Discovery

Article Title: Loss-of-function variants in ODAD1 disrupt ODA docking and induce actin cytoskeletal remodeling in primary ciliary dyskinesia

doi: 10.1038/s41421-026-00875-8

Figure Lengend Snippet: Video S22. Apical-out organoids-c.-41-2A-C+Lentivirus

Article Snippet: After the primers were annealed, the sgRNA was ligated into the lenti CRISPR V2 vector (Addgene, cat# 52961) using T4 DNA ligase.

Techniques:

Journal: bioRxiv

Article Title: PARG inhibition sequesters nuclear PAR-binding proteins, including XRCC1 and its partners, into nuclear condensates to elicit cytotoxicity

doi: 10.64898/2026.03.18.712393

Figure Lengend Snippet: A. V-abl B cells expressing Flag-tagged Cas9 were infected with a BFP-expressing lentiviral gRNA library. BFP+ cells were sorted and treated with doxycycline for five days to induce Cas9 expression. Indicated inhibitors treatment was performed at IC90 for six days. Then, DNA was collected from the cells for sequencing and analysis using the MAGeCK pipeline. B. Scatter plot of total CRISPR genes Z-scores for Niraparib and Olaparib IC90 treatments. Simple linear regression and Pearson correlation test were performed to determine R. C. Scatter plot of total CRISPR genes Z-scores for PDD0017273 and Olaparib IC90 treatments. Simple linear regression and Pearson correlation test were performed to determine R. D. Summary table of Pearson R coefficient for linear regression between cell lines sensitivity to PDD, olaparib, nirabarib and talazoparib. Depmap sensitivity score (PRISM repurposing data) of 536 cell lines to the indicated compound. Simple linear regression and Pearson correlation test were performed to determine R. Colored scaled is represented on the right and R coefficients are written in white. E. Heatmap representing z-score (color range) and False Discovery Rate FDR (size) for the genes targeting indicated in rows, in cells challenged with inhibitor indicated above, in columns. Gene implicated in homologous recombination are displayed. F. CRISPR screen z-scores ranking of co-essential genes with PDD00017273 treatment. SSBR related genes are shown in red, ADP-ribosylation related genes are shown in dark blue, HR related genes are shown in light grey.

Article Snippet: 100 million cells were infected with a BFP-expressing gRNA lentiviral library (Addgene Pooled Library #67988).

Techniques: Expressing, Infection, Sequencing, CRISPR, Homologous Recombination

Journal: bioRxiv

Article Title: TRIM9 switches the morphological phenotype of melanoma cells

doi: 10.64898/2026.03.17.712420

Figure Lengend Snippet: ( A ) Representative western blot of 1205Lu and WM266-4 lysates. GAPDH is used as a loading control. CRISPR/Cas9 generated TRIM9 −/− cells lack both short and long TRIM9 isoforms. ( B ) Representative images of 1205Lu TRIM9 +/+ and TRIM9 −/− cells plated on glass coverslips and stained for F-actin (phalloidin). ( C ) Cell area of 1205Lu cells on glass, TRIM9 −/− cells were larger than TRIM9 +/+ cells ( P =0.004, two-tailed t -test on log transformed data), n = 5 independent biological replicates (unique colors); n ≥ 20 cells per genotype per replicate, each dot indicates a cell. ( D ) Cell circularity on glass, TRIM9 +/+ cells were more circular (circle =1.0) ( P <.001, two-tailed t -test), n = 5 independent replicates; n ≥ 20 cells per genotype per replicate, each dot represents a cell. ( E ) 1205Lu cells plated on soft (~ 2 kPa) and stiff (~ 75 kPa) polyacrylamide hydrogels. ( F ) 1205Lu cell area on soft hydrogels, loss of TRIM9 increased cell area ( P <.001, two-tailed t -test), n = 3 independent biological replicates; n ≥ 39 cells per genotype, each dot indicates a cell. ( G ) Cell circularity on soft hydrogels; TRIM9 +/+ cells are more circular than TRIM9 −/− cells ( P =0.03, two-tailed t -test), n = 3 independent biological replicates-unique colors; n ≥ 39 cells per genotype per replicate, each dot indicates a cell. ( H ) Time spent in a bleb-like state on soft hydrogels; TRIM9 +/+ cells spend more time blebbing ( P =.04, two-tailed t -test on log transformed data), n = 3 independent biological replicates; n ≥ 39 cells per genotype, each dot indicates a cell. ( I ) Phalloidin stained 1205Lu cells plated on glass for two hours. ( J ) Cell area for cell spreading; TRIM9 −/− cells exhibit increased spreading at two hours as compared to TRIM9 +/+ ( P <.001, two-tailed t -test on natural log transformed data), n = 3 independent biological replicates; n ≥ 100 cells per genotype per replicate, each dot represents a cell.

Article Snippet: WM266-4 TRIM9 −/− cells were generated by CRISPR/Cas9 lentiCRISPR V2 (Addgene #52961) using gRNA targeting the sequence 5’-GCG GCT ATG GCT CCT ACG GGG GG-3’ in exon 1 of the human TRIM9 gene.

Techniques: Western Blot, Control, CRISPR, Generated, Staining, Two Tailed Test, Transformation Assay