cas9 expressing lentivirus  (Addgene inc)

Journal: Leukemia

Article Title: Identifying targeted therapies for CBFA2T3::GLIS2 acute myeloid leukemia

doi: 10.1038/s41375-025-02845-7

Figure Lengend Snippet: A Diagram of genome-scale CRISPR-Cas9 drop-out screen. B Dotplot showing JAK2 dependency scores across 24Q4 DepMap cell lines ( n = 1178) and WSU-AML (drop-out CRISPR screen). Dots represent AML CBFA2T3::GLIS2 -positive cell lines (red), AML CBFA2T3::GLIS2 -negative cell lines (black), and all other cell lines (gray). JAK2 gene effect dependency score computed based on the Chronos algorithm for the 24Q4 CRISPR cell lines and based on the hypergeometric test as average fold change lognorm day 21 vs day 0 for WSU-AML. Significance: Chronos gene effect < −0.5 for CMS and M-07e, and average LFC gene effect < −1.5, P -value < 0.05 for WSU-AML. C Venn diagram showing the overlap between non-common essential dependencies in the CBFA2T3::GLIS2 AML cell lines CMS, M-07e (24Q4 DepMap) and WSU-AML (drop-out CRISPR screen). Overlap significance estimated based on the two-tailed Fisher’s exact test. **** P < 0.0001. D Western immunoblot showing the effect of JAK2 knock-out using two different sgRNA (sgJAK2-1 and sgJAK2-2) after 4 days of doxycycline treatment in M-07e cell line; actin was used as a loading control. E Competitive growth assays demonstrating that JAK2 is essential for cell growth in the three CBFA2T3::GLIS2 AML cell lines. Cells were transduced with nontargeting sgRNA (sgNT) and JAK2 sgRNA (sgJAK2-1 and sgJAK2-2) vectors that co-express GFP. Cell growth was evaluated in vitro by the change in percentage of GFP+ cells post doxycycline induction. Shown are mean ± SD, n = 3. P -value calculated using a 2-way ANOVA with multiple comparisons test (comparison to sgNT). * P < 0.05; *** P < 0.001; **** P < 0.0001. F Cumulative cell growth measured using an ATP-based CellTiter-Glo assay in AML cells either transduced with sgNT or sgJAK2 after FACS-sort for GFP-positive cells. Results represented as mean ± SD ( n = 8). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, determined by two-way ANOVA test followed by Dunnett’s multiple comparisons test. Shown is a representative of three experiments. G Flow cytometry analysis of Annexin V+ and Annexin V+/PI+ in AML cells expressing the indicated sgRNAs at day 7 after doxycycline induction in WSU-AML cell line. Shown are means ± SD, n = 2; experiment performed three times. P -value calculated using a 2-way ANOVA test (comparison to sgNT). The percentage of Annexin⁺/PI⁻ and Annexin⁺/PI⁺ cells was increased in JAK2 KO cells compared with sgNT (sgJAK2-1 P = 0.0017 and sgJAK2-2 P < 0.0001 for Annexin⁺/PI⁻; sgJAK2-1 P = 0.0167 and sgJAK2-2 P < 0.0001 for Annexin⁺/PI⁺). The percentage of Annexin - /PI + was not significantly difference in sgJAK2-1 vs. sgNT and sgJAK2-2 vs. sgNT.

Article Snippet: AML and PDX cells were transduced with lentivirus co-expressing Cas9 and mCherry (a gift from Marco Herold, Addgene plasmid # 70182; RRID: Addgene_70182).

Techniques: CRISPR, Two Tailed Test, Western Blot, Knock-Out, Control, Transduction, In Vitro, Comparison, Glo Assay, Flow Cytometry, Expressing

Journal: Leukemia

Article Title: Identifying targeted therapies for CBFA2T3::GLIS2 acute myeloid leukemia

doi: 10.1038/s41375-025-02845-7

Figure Lengend Snippet: A Diagram of genome-scale CRISPR-Cas9 screen for ruxolitinib modifier genes. B Correlation between the differential log2 fold change in sgRNA abundance between DMSO and ruxolitinib for the two cell lines screened (CMS and WSU-AML) after 14 days of treatment with ruxolitinib. Hits that confer resistance to ruxolitinib are shown in red. All other genes are in gray. C Guide level log2 fold changes for normalized data relative to plasmid pool DNA for the gene RASA2 in the CMS cell line. Each sgRNA is represented. P-values calculated using the hypergeometric test for significant difference in the screen. Shown are the means ± SD, n = 2. ** P < 0.01; **** P < 0.0001. D Western immunoblot showing the effect of RASA2 KO using two different sgRNA (sgRASA2-1 and sgRASA-2) in the CMS cell line; vinculin was used as a loading control. E CMS cells were infected with a nontargeting control (sgNT) (gray) and an sgRNAs targeting RASA2 (red). Non-infected control cells (NIC) were represented in black. Bar graphs showing cell numbers after 7 days of cell culture with ruxolitinib 150 nM (Ruxo) or DMSO. Shown are the means ± SD ( n = 3). P -values calculated using a 2-way ANOVA test. ** P < 0.01; ns not significant. F Cell viability assay in CMS cells transduced with sgNT or sgRASA2 puromycin selection vector. Cells were treated with ruxolitinib for 72 h and viability evaluated using the CellTiter-Glo assay. Line graphs represent means ± SD ( n = 3). AUC values for sgNT and sgRASA2 (guides 1 and 2) were compared using Mann–Whitney unpaired t -test. * P < 0.05. G Cell viability assay in CMS cells transduced with sgNT or sgRASA2 puromycin selection vector or untransduced. Cells were treated with tofacitinib for 72 h and viability evaluated using CellTiter-Glo assay. Line graphs represent means ± SD ( n = 3). AUC values for sgNT and sgRASA2 (guides 1 and 2) were compared using Mann–Whitney unpaired t -test. * P < 0.05.

Article Snippet: AML and PDX cells were transduced with lentivirus co-expressing Cas9 and mCherry (a gift from Marco Herold, Addgene plasmid # 70182; RRID: Addgene_70182).

Techniques: CRISPR, Plasmid Preparation, Western Blot, Control, Infection, Cell Culture, Viability Assay, Transduction, Selection, Glo Assay, MANN-WHITNEY

Journal: medRxiv

Article Title: Fusion oncoproteins and cooperating mutations define disease phenotypes in NUP98 -rearranged leukemia

doi: 10.1101/2025.01.21.25320683

Figure Lengend Snippet: A . Experimental schema of induction of cooperating alterations ( RB1 , WT1 ) in cbCD34/Cas9 models. B . Cell growth assays of cbCD34/Cas9 NUP98::KDM5A with gRNAs targeting the AAVS, RB1 , or WT1 loci ( left ), and cytospin of cells on day 35 ( right ). C . Induction rates of indel (insertions and deletions) at day 4 and 39 in each condition. Bars represent fractions of indel rates in all target sequence reads, and dots represent out-of-frame indel ratio among total indels. D . Flow gating ( left ), CD34+ CD41a+ positivity ( mid ), and CD34+ CD41a- ( right ) among mCherry+ GFP+ mAmetrine+ live cells. E . PCA of RNAseq of gRNA-transduced NUP98::KDM5A cbCD34 models at day 35. F . DEG analysis between AAVS controls and RB1 -gRNA conditions ( left ) and gene ontology (GO) term analysis of DEGs ( right ). Colors indicate DEGs and GO terms (red: high in RB1 -gRNA conditions, blue: low in RB1 -gRNA conditions) . G . DEG analysis between AAVS controls and WT1 -gRNA conditions as shown in F . H . UMAP plots of scRNAseq data from gRNA-transduced NUP98::KDM5A cbCD34 models at day35, showing marker gene expression ( left ), annotated clusters ( mid ), and cell distributions among conditions ( right ). Colors in plots indicate relative expression levels, clusters, and cell density, respectively. I . Enrichment of cells with each cluster indicated by colors and sizes. J . Pseudotime along myeloid (HSC→GMP→monocytes) and platelet (HSC→MEP→MK→platelet) trajectories. Colors represent pseudotime scores of each single cell inferred by Slingshot. K . RB1 ( top ) and CDKN2A ( bottom ) expression along the pseudotime axis in each condition with red curves show average expressions. L . DEG analysis between the platelet-like and MK-like clusters in the AAVS-control condition ( left ) and GO term analysis ( right ) of genes high in the platelet-like cluster (red) and the MK-like cluster (blue). M. Schematics illustrating platelet differentiation in normal hematopoiesis and NUP98::KDM5A models. Assay data was obtained in technical triplicates from an established NUP98::KDM5A/Cas9 line and independent experiments. One data point in C was not obtained due to technical errors. RNAseq data was obtained from six independent experiments. In B-D , statistical tests were performed by linear mixed effect model ( B ) or two-sided Student’s t-test by comparing day 4 and day 39 ( C ) or gRNA conditions and AAVS controls ( D ), and limma ( F , G ) followed by the Benjamini-Hochberg adjustment when applicable. DEGs in scRNAseq ( L ) were identified using FindMarker function in Seurat package with default settings, which calculate adjusted P -values with limma implementation of the Wilcoxon rank-sum test followed by Bonferroni correction. Asterisks indicating P -values or adjusted P -values <0.05. Error bars indicate mean ± s.e.m.

Article Snippet: N-terminal HA tags (YPYDVPDYA) were introduced by amplification during cloning. cDNA encoding Cas9 expressing lentivirus vector (Addgene, FUCas9GFP, Plasmid #85555) and used without cloning. gRNA expressing vector were established by Center for Advanced Genome Engineering (CAGE) in St. Jude Children’s Research Hospital using validated gRNAs and LentiGuide-Ametrine backbone expressing mAmetrine under mPGK promoter as described previously .

Techniques: Sequencing, Marker, Expressing, Control