Journal: bioRxiv

Article Title: High-throughput Genome Wide CRISPR Knock Out mechanical sort identifies genes driving metastatic cancer cell softening

doi: 10.64898/2026.02.12.705447

Figure Lengend Snippet: Cancer cells expressing Cas9 are transduced with a lentiviral library containing sgRNAs across the entire protein-coding genome at a low MOI to allow for single gene knockouts. Antibiotic selection ensures only cells with gene knockouts are included in mechanical screen. Next, the library of cells with single gene knockouts are processed with a microfluidic stiffness-based cell sorting device into 5 mechanical subsets. The inset shows a side and top view of the ridged region of the device where cells must deform underneath each diagonal ridge. Softer cells deform easily underneath the ridges and follow hydrodynamic streamlines to provide a slight negative deflection towards outlets 1 and 2. Stiffer cells resist deformation and are deflected along the diagonal ridge resulting in trajectories towards outlets 4 and 5. Genomic DNA is harvested from each mechanical subset, the sgRNA regions are amplified and sequenced, and the distribution of sgRNAs before and after the mechanical screen is used to determine the correlation of gene knockouts with mechanical subsets.

Article Snippet: The human CRISPR Brunello lentiviral pooled library was designed to optimize on-target activity and reduce off-target effects (Addgene 73178-LV) .

Techniques: Expressing, Transduction, Selection, FACS, Amplification

Journal: Science Advances

Article Title: Senataxin and DNA-PKcs redundantly promote non-homologous end joining repair of DNA double strand breaks during V(D)J recombination

doi: 10.1126/sciadv.ads5272

Figure Lengend Snippet: ( A ) Schematic of the retroviral V(D)J recombination substrate pMG-INV. The open and filled triangles represent the RSSs. The retroviral LTRs, CEs, and SEs upon RAG cleavage and CJs and SJs after NHEJ-mediated repair are indicated. The anti-sense GFP cDNA is inverted to the sense orientation upon completion of the recombination and its expression driven by the retroviral LTR (green). ( B ) Schematic diagram of a genome-wide CRISPR-Cas9 screen in WT abl pre-B cells for identifying genes important for V(D)J recombination upon DNA-PKcs inhibition with NU7441. ( C ) Fold enrichments of selected gRNAs from screens conducted in DMSO and NU7441-treated WT abl pre-B cells (both also treated with imatinib). The fold enrichment is calculated as the ratio of the normalized read counts of a gRNA from GFP − cells over that from GFP + cells. Asterisk denotes (*) five gRNAs to each gene. ( D ) Flow cytometric analysis for GFP expression in WT and three independently isolated Setx −/− abl pre-B cell lines with pMG-INV and treated with imatinib (imat.) in the presence or absence of the DNA-PKcs kinase inhibitor NU7441 for the indicated times. The percentages of GFP + cells are indicated in the top left corners of the histograms.

Article Snippet: For each screen, ~180 × 10 6 cells were transduced with a lentiviral gRNA library containing 90,230 gRNAs targeting 18,424 mouse genes (Addgene, #67988) at a 40 to 50% transduction efficiency, as determined by the percentage of blue fluorescent protein (BFP)–positive cells after transduction.

Techniques: Retroviral, Expressing, Genome Wide, CRISPR, Inhibition, Isolation