GSE135300
GSE GEONext Generation Sequencing: in vivo genome-wide CRISPR sgRNA screen in primary cancer-initiating and propagating bcCML stem cells
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Summary
While Chronic Myelogenous Leukemia (CML) is generally well controlled with Imatinib and other kinase inhibitors, blast crisis CML (bcCML) continues to be resistant to current therapies and remains highly lethal. Here we report a genome-wide in vivo CRISPR screen to better define the biological determinants of bcCML establishment and propagation in a physiologic context. This screen identified a large number of new genes and programs critically required for bcCML including those essential for chromatin remodeling, spliceosomal assembly, PLK1 and Myc signaling.
Overall Design
We generated lentiviral particles from the Brie library (Addgene #73633) and infected 300 million freshly isolated Cas9-GFP+ Lineage- blast crisis CML (bcCML) stem cells from the spleen of pre-morbid mice at an MOI of 0.25 such that a single cell is infected with only one virus carrying one gRNA. 60 hours post spinfection, an aliquot of cells was collected and represented a pool of infected cells with about 200 cells/guide (Pre-Sel sample). The cells were then selected for 48h in puromycin to enrich for cells carrying gRNAs (Post-Sel sample). 35 million cells were transplanted in sub-lethally irradiated recipients (1 million cells/mouse) to ensure a coverage of >400 cells/guide. The recipient mice were sacrificed 7 days post-transplant, before the onset of full-blown disease. The cells from the spleen of all 35 mice were pooled into one sample (InV) after sorting for equal numbers (~1.1 million cells/mouse) of leukemic cells.
Analysis (2 steps)
View Data Processing- Reads were counted by first searching for the CACCG sequence in the primary read file that appears in the vector 5â to all sgRNA inserts.
- The next 20 nts are the sgRNA insert, which was then mapped to a reference file of all possible sgRNAs present in the library.
Supplementary Files (1)
GEO Samples (3)
SRA Experiments (3) and Runs (3)
Total: 4435 MBSample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR9890532 | 53253839 | 3994037925 | 1531.51 | BeS.fastq.gz, SRR9890532, SRR9890532.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR9890533 | 49462425 | 3709681875 | 1419.88 | AfS.fastq.gz, SRR9890533, SRR9890533.sralite | SRA |
Sample attributes
Original files (1)
Runs (1)
| Run | Spots | Bases | Size (MB) | Files | Link |
|---|---|---|---|---|---|
| SRR9890534 | 51933817 | 3895036275 | 1483.42 | InV.fastq.gz, SRR9890534, SRR9890534.sralite | SRA |