genome wide crispr cas9 knockout library screen  (Addgene inc)

Journal: bioRxiv

Article Title: Simultaneous single-cell CRISPR, RNA, and ATAC-seq enables multiomic CRISPR screens to identify gene regulatory relationships

doi: 10.1101/2025.02.11.637716

Figure Lengend Snippet: Pooled CRISPRi screen identifies gene knockdowns which modulate resistance to dasatinib A. PCA plot showing reproducibility of replicate samples from the genome-wide screen. B. Volcano plot showing gene knockdowns exhibiting sensitizing and desensitizing phenotypes. Blue dots represent all significant hits. Genes highlighted in red were previously known to affect dasatinib sensitivity. C. Schematic showing overview of the arrayed validation experiment conducted to validate hits from the primary genome-wide screen. D. Bar plot displaying results from the arrayed validation experiment showing percent increase in cell death for each of the guide expressing cells over non-targeting control guide expressing cells. Error bars represent standard deviation. E. GO analysis for the 60 genes included in the arrayed validation experiment.

Article Snippet: K562 cells containing dCas9-KRAB CRISPRi machinery were infected with CRISPRi V2 library containing the top5 guides (Addgene 83969) to achieve 1000-fold coverage at an MOI of 0.3.

Techniques: Genome Wide, Expressing, Control, Standard Deviation

Journal: bioRxiv

Article Title: Simultaneous single-cell CRISPR, RNA, and ATAC-seq enables multiomic CRISPR screens to identify gene regulatory relationships

doi: 10.1101/2025.02.11.637716

Figure Lengend Snippet: Flow cytometry gating strategy Gating strategy for flow cytometry data of k562/CRISPRi cells to study the effect of dasatinib on CRISPRi constructs targeted against HIC-2, PTH2R, and ZFPM2 stained with 1ug/ml EthD-1 and 2 μM calcein-AM analyzed by flow cytometry. The first panel shows all events with a gate (population 1) for cells. The second panel shows population 1 with a gate for singlets (population 2). The third panel shows Quarter 1 = Green or BL1-A for live cells and Quarter 3 = Red or YL2-A for dead cells (population 3).

Article Snippet: K562 cells containing dCas9-KRAB CRISPRi machinery were infected with CRISPRi V2 library containing the top5 guides (Addgene 83969) to achieve 1000-fold coverage at an MOI of 0.3.

Techniques: Flow Cytometry, Construct, Staining

Journal: bioRxiv

Article Title: Simultaneous single-cell CRISPR, RNA, and ATAC-seq enables multiomic CRISPR screens to identify gene regulatory relationships

doi: 10.1101/2025.02.11.637716

Figure Lengend Snippet: ZFPM2 and PTH2R are necessary and sufficient for increased resistance to dasatinib. A. qPCR data showing CRISPRi mediated knockdown of gene expression for the genes HIC2, PTH2R and ZFPM2. B. Flow-cytometry plots at 72h post dasatinib treatment showing the distribution of cells for each of the three gene knockdown conditions along with control cells treated with dasatinib and stained with Calcien-AM and EthD-1 to identify live and dead cells respectively. C. Timecourse experiment showing flow-cytometry data from 72h to 168h post dasatinib treatment quantifying % viable cells in HIC2 KD, ZFPM2 KD and control cells. Each data point represents the mean ± SD of three biological replicates (n=3). Statistical significance was assessed at each time point using unpaired two-tailed t-tests comparing HIC2 KD or ZFPM2 KD vs. NTC. Black asterisks indicate significant differences for HIC2 KD vs. NTC, while purple asterisks indicate significant differences for ZFPM2 KD vs. NTC (* corresponds to p < 0.05, ** corresponds to p < 0.01 and *** corresponds to p < 0.001).

Article Snippet: K562 cells containing dCas9-KRAB CRISPRi machinery were infected with CRISPRi V2 library containing the top5 guides (Addgene 83969) to achieve 1000-fold coverage at an MOI of 0.3.

Techniques: Knockdown, Expressing, Flow Cytometry, Control, Staining, Two Tailed Test