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

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: bioRxiv

Article Title: Massively parallel reporter assays identify functional enhancer variants at QT interval GWAS loci

doi: 10.1101/2025.03.11.642686

Figure Lengend Snippet: ( Left ) Workflow for variant selection for the MPRA experiment. ( Center ) Schematic of the MPRA oligo design and library cloning process. ( Right ) Workflow describing the steps for transfection, sequencing, and computational analysis.

Article Snippet: The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility.

Techniques: Variant Assay, Selection, Cloning, Transfection, Sequencing

Journal: bioRxiv

Article Title: Massively parallel reporter assays identify functional enhancer variants at QT interval GWAS loci

doi: 10.1101/2025.03.11.642686

Figure Lengend Snippet: (A) Relative luciferase activity (Firefly/Renilla) for 11 randomly selected DA variants are shown. Two sample t-tests between the reference and alternative (variant) alleles were performed to measure statistical significance. *** FDR< 0.0001; ** FDR < 0.001; * FDR < 0.01. (B) MPRA fold change measured for 11 variants are compared with those measured by luciferase assays. Each variant is color coded by their -log10(FDR) value in luciferase assays.

Article Snippet: The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility.

Techniques: Luciferase, Activity Assay, Variant Assay

Journal: bioRxiv

Article Title: Massively parallel reporter assays identify functional enhancer variants at QT interval GWAS loci

doi: 10.1101/2025.03.11.642686

Figure Lengend Snippet: (A) Bar plots show the numbers of MPRA (top), EA (middle), and DA (bottom) variants, grouped by loci. (B) For the NOS1AP locus, variants from QT interval GWAS datasets (GWAS2014 from and GWAS2022 from ) and eQTL variants for five different genes ( NOS1AP, C1orf226, ATF6, OLFML2B, SH2D1B ) from the GTEx project’s heart left ventricles are shown. For eQTL analysis, variants were limited to the MPRA test variants. MPRA (blue), EA (green), and DA (red) variants are shown at the bottom. DA variants are highlighted as triangles with dashed lines in all tracks. (C) The distribution of Pearson correlation of normalized gene expression between NOS1AP and all other genes is shown. Its correlation with C1orf226 is an outlier and is indicated by a red arrow.

Article Snippet: The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility.

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Massively parallel reporter assays identify functional enhancer variants at QT interval GWAS loci

doi: 10.1101/2025.03.11.642686

Figure Lengend Snippet: (A) Effect sizes estimated by eQTLs (X-axis) and aseQTLs (Y-axis) are compared for GTEx heart left ventricles datasets. All variants tested for the genes in the 31 QT interval loci are used for comparison. Blue circles represent significant eQTL variants. (B) A Venn diagram shows the overlaps of DA variant-gene pairs identified by three different methods. (C) For the CNOT1 locus, variants from the two QT interval GWAS datasets and aseQTLs for eight different genes from GTEx heart left ventricles are shown as genomic tracks. For aseQTLs, variants are restricted to the MPRA test variants. MPRA (blue), EA (green), and DA (red) variants are shown at the bottom. DA variants are highlighted as triangles with dashed lines in all tracks. (D) A heatmap summary of all DA variant-gene pairs identified by at least one method. The variants are grouped by locus. Variants missing in the corresponding data sets are highlighted.

Article Snippet: The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility.

Techniques: Comparison, Variant Assay

Journal: bioRxiv

Article Title: Massively parallel reporter assays identify functional enhancer variants at QT interval GWAS loci

doi: 10.1101/2025.03.11.642686

Figure Lengend Snippet: The -log10 transformed P-values of GWAS and eQTL/aseQTL are compared across MPRA and GWAS tested variants for the genes in five different loci: NOS1AP (A) , CNOT1 (B) , SCN5A (C) , PLN (D) , and KCNQ1 (E) . Only the genes associated with DA variants with significant eQTL or aseQTL are included in this analysis. High confidence genes are highlighted in red for each locus.

Article Snippet: The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility.

Techniques: Transformation Assay