2016 mouse genome wide crispr guide rna library v2  (Addgene inc)

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: Genome-wide knockout CRISPR screens define the common and differential fitness genes in the 4T1 and 4T07 cells. A, Schematic outline for the performed screens’ pipeline. B, Enumeration of the revealed cell line–specific and common (core) fitness genes. C, Ranked differential gene fitness score between the 4T1 and 4T07 cells. A value >0 denotes a 4T1-specific fitness gene; a value <0 denotes a 4T07-specific fitness gene. D, Schematic representation integrating the 4T1-specific fitness genes (green) and proliferation suppressor genes (positively selected genes; gray) in the canonical PI3K-AKT pathway. E and F, Gene set enrichment analysis comparing the enrichment of genes previously found to be induced by AKT upregulation ( E ) or PTEN downregulation in the two cell lines ( F ). NES, normalized enrichment scale.

Article Snippet: The mouse Genome-Scale CRISPR Knockout (mGeCKO) library A (Addgene, #1000000052), composed of ∼68,000 guide RNAs (gRNA): 3 gRNAs/gene, 4 gRNAs/miRNA, and 1,000 nontargeting sequences, was amplified and prepared for next-generation sequencing (NGS; MiSeq Illumina) to assess the gRNA distribution and library quality, as previously described ( ).

Techniques: Genome Wide, Knock-Out, CRISPR

Journal: Cancer Research

Article Title: Targeting the Dependence on PIK3C3-mTORC1 Signaling in Dormancy-Prone Breast Cancer Cells Blunts Metastasis Initiation

doi: 10.1158/0008-5472.CAN-23-2654

Figure Lengend Snippet: Pik3c3 is a therapeutic vulnerability of dormant cancer cells in a HER2 breast cancer model. A, Schematic for the MMTV-rtTA;TetO-Her2 and the origin of the primary and recurrent lines. doub, population doubling; dox, doxycycline; T0, reference time point; T-End, endpoint. B, Schematic for the CRISPR screen performed in the primary and recurrent MMTV-rtTA;TetO-Her2 cells. C, Graphs demonstrating the identification of Pik3c3 as a recurrent cell-specific fitness gene, as defined by the FDR and fold change of its gRNA(s) in the screens’ endpoint in reference to T0 (see Supplementary Methods for details). D, Venn diagram highlighting the number of identified recurrent cell-exclusive fitness genes. E, Cell viability assay comparing the sensitivity of primary and recurrent MMTV-rtTA;TetO-Her2 cells to VPS34IN1. AUC was quantified for each line and all lines’ sensitivity to VPS34IN1 was statistically different. F, Schematic for a cell death assay investigating the effect of VPS34IN1 (10 μmol/L) on the 54074 primary cells either during proliferation (in prescence of doxycycline) or dormancy (cells deprived of doxycycline). G, Quantifications of the percentage of dead 54074 cells after 24 and 48 hours treatment with VPS34IN1.

Article Snippet: The mouse Genome-Scale CRISPR Knockout (mGeCKO) library A (Addgene, #1000000052), composed of ∼68,000 guide RNAs (gRNA): 3 gRNAs/gene, 4 gRNAs/miRNA, and 1,000 nontargeting sequences, was amplified and prepared for next-generation sequencing (NGS; MiSeq Illumina) to assess the gRNA distribution and library quality, as previously described ( ).

Techniques: CRISPR, Viability Assay