cdna encoding cas9 expressing lentivirus vector  (Addgene inc)

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