primary antibody mouse monoclonal iggik anti cas9 ab 7a9 3a3  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: FDXR Upregulation by p63/p73 is a Prognostic and Therapeutic Marker of HTLV-1-Associated Adult T Cell Leukemia/Lymphoma

doi: 10.64898/2026.02.25.707207

Figure Lengend Snippet: (A,B) RNA-Seq data retrieved from Protein Atlas were stratified by FDXR expression (≥75 nTPM = Mid-high_FDXR, <75 nTPM = Mid-low_FDXR). Differential transcription factor (TF) activity between groups was inferred using VIPER , based on regulatory networks constructed with DoRothEA and ARACNe. The bubble plot in (B) ranks TFs based on their differential activity in the two groups. The colour of the dots reflects relative TF activity, while the size of the dots represent -Log10 adjusted p values (FDR) calculated with the limma package. (C–F) Expression of TAp63 mRNA (C), p63α protein (D), TAp73 mRNA (E) and p73 protein (F) in HTLV-1 + , ATL, and uninfected cell lines and primary cells. Relative mRNA expression was quantified by qPCR; protein expression was assessed by western blot. In panels (C,E), each dot represents an independent qPCR assay (n = 3), and data are presented as mean ± SD. Relative mRNA levels were calculated by ΔΔCq using TBP expression as reference. Data were analyzed by non-parametric Wilcoxon rank-sum test. (G,H) Relative expression of TAp63 (G) and TAp73 (H) mRNA in primary CD4 T cells from PLHTLV stratified by disease condition and healthy controls. Relative mRNA expression was quantified by qPCR using the ΔΔCq method and TBP expression as reference. Each dot represents a donor and data are depicted as median ± IQR. Data were analyzed by non-parametric Kruskal–Wallis test followed by Dunn’s post hoc test with Benjamini–Hochberg correction for multiple comparisons. (I,N) Effect of TP63 and TP73 knockout on FDXR expression. The HTLV-1–infected MT-4 cell line (I-K) and the ATL-derived ATL-55T cell line (L-N) were stably transfected with Cas9 and subsequently transduced with lentiviruses expressing either a non-targeting (NT) sgRNA or sgRNAs targeting TP63 or TP73. Stable knockout cell lines were lysed and used to assess protein expression by Western blot. Panels I, L show p63 and FDXR expression in MT-4 and ATL-55T cells, respectively. Panels J, M show p73 and FDXR expression in MT-4 and ATL-55T cells, respectively. Wild-type and non-transduced Cas9-expressing cells served as additional controls. Panels K, N show relative FDXR protein levels normalized using actin as a loading control and the NT sgRNA condition as reference level. Barplots in K, N show mean ± SD for each condition. N = 2. **** p <0.0001; ** p <0.01; *p <0.05.

Article Snippet: The primary antibodies used included anti-FDXR (1:1000, Rabbit, Proteintech #15584-1-AP), anti-p53 (Rabbit, Cell Signaling #2527), anti-p63α (1:1000, Rabbit, Cell Signaling #13109), anti-p73 (1:1000, Rabbit, Cell Signalling #14620), anti-S. pyogenes-Cas9 (1:1000, Mouse, Cell Signalling #14697) anti-VDAC (1:500, Rabbit, Invitrogen #MA5-33205), anti-Tax (1:250, Mouse, Merck #MABF3063), anti-HTLV-1-p24 (1:250, Mouse, Santa-Cruz #sc-53891), anti-HTLV-1-gp46 (1:250, Mouse #sc-53890), anti-alpha Tubulin (1:15000, Rabbit, GeneTex #GTX112141), anti-beta Actin (1:15000, Mouse, Cell Signaling #3700).

Techniques: RNA Sequencing, Expressing, Activity Assay, Construct, Western Blot, Knock-Out, Infection, Derivative Assay, Stable Transfection, Transfection, Transduction, Control

Journal: bioRxiv

Article Title: Rational design of synthetic proteins using a genome-scale CRISPR screen

doi: 10.64898/2026.02.19.706875

Figure Lengend Snippet: a , Human K562-EGFP cells were lentivirally transduced with CRISPRa (dCas9-VP64 + p65-HSF1) and a CRISPRa guide RNA (gRNA) library targeting ∼19,000 genes, selected, and nucleofected with a GFP-targeting Cas9 ribonucleoprotein complex and single-stranded repair template to convert GFP to BFP. Cells are flow sorted based on genome repair outcomes, and gRNA representation is computed in sorted bins for homology-directed repair (HDR) and non-homologous end-joining (NHEJ). b , Flow cytometry gating for repair outcomes (HDR or NHEJ) and quantification. c , Normalized enrichment in the HDR population for the four most enriched CRISPRa gRNAs targeting the indicated genes. The fold-change of each gRNAs is shown as blue lines and the average is indicated by the diamond. d , Gene ranks and significance for HDR enrichment with robust rank aggregation (RRA) using the four most enriched gRNAs. The highlighted region indicates genes with FDR-adjusted RRA P adj < 0.1. e , RNA expression in K562 cells of the top enriched HDR genes (FDR P adj < 0.1).

Article Snippet: The membranes were then incubated overnight at 4 °C with one of the following primary antibodies: mouse anti- Sp Cas9 (Cell Signaling 14697, 1:2000), rabbit anti-TOP3A (Proteintech 14525-1-AP, 1:2000), rabbit anti-RMI1 (Proteintech 14630-1-AP, 1:500), or rabbit anti-ACTB (Proteintech 81115-1-RR) in 5% BSA (VWR AAJ65097) dissolved in TBS-T.

Techniques: Transduction, Non-Homologous End Joining, Flow Cytometry, RNA Expression

Journal: bioRxiv

Article Title: Rational design of synthetic proteins using a genome-scale CRISPR screen

doi: 10.64898/2026.02.19.706875

Figure Lengend Snippet: a , Candidates for protein fusion with Cas9 nuclease for improved precise gene editing. b , Precise repair (GFP-to-BFP conversion) for 20 TruEditors as measured by flow cytometry. TruEditor constructs were nucleofected into human kidney HEK293-EGFP cells with a GFP-targeting gRNA and a single-stranded repair template encoding BFP-specific edits. Points represent three independent transfection replicates and the diamond indicates the mean. Grey rectangle represents the 95% confidence interval for the Cas9 control. The heatmap shows the length of fused proteins. c , Comparison of TruEditors with significant increases in precise repair as direct Cas9 fusions (XTEN linker) versus co-expression (2A cleavage). Points represent the mean of three replicates with SEM error bars. d , Precise install of the protective G127V PRNP variant in HEK293 cells. e , Precise install of the sickle cell disease-causative HBB E7V variant in HEK293 cells. f , Precise knock-in of FLAG protein affinity tag at the N terminus of the PARP1 gene in HEK293 cells. g , Representative immunofluorescent images of nuclear 53BP1 foci in human lung A549 cells expressing the indicated gene editor or treated with the NHEJ inhibitor M3814 (2 μM) or vehicle (DMSO) control. Cells were either treated for 4 hours with vehicle, doxorubicin (0.5 μM), or doxorubicin and then allowed to recover for 24 hours post-washout. Scale bar, 10 μm. h , Recovery of nuclear 53BP1 foci after doxorubicin treatment in A549 cells. i , Comparison of TruEditors and prime editing to correct the MUT R369H variant in HEK293 cells. j , Precise correction of MUT R369H variant in HEK293 cells. k , Precise correction of the lung cancer driver KRAS G12S mutation in A549 cells. l , Precise correction of the atypical teratoid/rhabdoid tumor (ATRT) driver SMARCB1 M27fs mutation in human brain BT16 cells. For all comparisons, experiments had at least 3 independent replicates, and significance was determined using a one-way ANOVA. Post-hoc pairwise comparisons were conducted using Tukey’s HSD test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The membranes were then incubated overnight at 4 °C with one of the following primary antibodies: mouse anti- Sp Cas9 (Cell Signaling 14697, 1:2000), rabbit anti-TOP3A (Proteintech 14525-1-AP, 1:2000), rabbit anti-RMI1 (Proteintech 14630-1-AP, 1:500), or rabbit anti-ACTB (Proteintech 81115-1-RR) in 5% BSA (VWR AAJ65097) dissolved in TBS-T.

Techniques: Flow Cytometry, Construct, Transfection, Control, Comparison, Expressing, Variant Assay, Knock-In, Mutagenesis

Journal: bioRxiv

Article Title: Rational design of synthetic proteins using a genome-scale CRISPR screen

doi: 10.64898/2026.02.19.706875

Figure Lengend Snippet: a , AlphaFold3 structure predictions for the indicated, full-length Cas9 fusion TruEditors. b , Linear representation of annotated protein domains from the indicated proteins. Cloned domains or combinations of domains are indicated as horizontal lines and their lengths given in amino acids. c , Precise repair (GFP-to-BFP editing) rates for the indicated minimal or full-length TruEditors. TruEditor constructs were nucleofected into HEK293-EGFP cells with a GFP-targeting gRNA and a single-stranded repair template encoding BFP-specific edits. Points represent three independent transfection replicates, and the diamond indicates the mean. Grey rectangle represents the 95% confidence interval for the Cas9 control.

Article Snippet: The membranes were then incubated overnight at 4 °C with one of the following primary antibodies: mouse anti- Sp Cas9 (Cell Signaling 14697, 1:2000), rabbit anti-TOP3A (Proteintech 14525-1-AP, 1:2000), rabbit anti-RMI1 (Proteintech 14630-1-AP, 1:500), or rabbit anti-ACTB (Proteintech 81115-1-RR) in 5% BSA (VWR AAJ65097) dissolved in TBS-T.

Techniques: Clone Assay, Construct, Transfection, Control

Journal: bioRxiv

Article Title: Rational design of synthetic proteins using a genome-scale CRISPR screen

doi: 10.64898/2026.02.19.706875

Figure Lengend Snippet: a , Affinity proteomics (AP) to identify protein-protein interactomes for different TruEditors. FLAG-tagged TruEditors were immunoprecipitated from HEK293 cells after 24 hours and interacting proteins were identified on a Orbitrap Eclipse Tribrid mass spectrometer. b , Pearson correlation of peptide spectral match (PSM) counts ( n = 2 - 3 biological replicates per TruEditor). c , Protein-protein interaction networks for TruEditors and their significant interactors (average PSM fold-change over Cas9 alone > 10 and P adj < 0.05). Edge thickness (average PSM) and color (significance analysis of interactome [SAINT] score ) correspond to the strength of the connection. Nodes are colored by Gene Ontology category. d , Ternary complex of the Cas9- BLM 2-194 TruEditor with AP-validated interactors TOP3A and RMI1 as predicted by AlphaFold3. Key BLM 2-194 interaction residues at the interface with TOP3A ( light grey ) and RMI1 ( medium grey ) are highlighted in pink . e , Co-immunoprecipitation of FLAG-tagged Cas9 only, Cas9- BLM 2-194 and different Cas9- BLM 2-194 mutants (N8A, F35A, N8A/F35A, ΔA2-L21, ΔA2-T41) in HEK293 cells. f , Precise editing rates (GFP-to-BFP editing) for Cas9 only, Cas9- BLM 2-194 and Cas9- BLM 2-194 mutants in HEK293-GFP cells as measured by flow cytometry ( n = 3 biological replicates).

Article Snippet: The membranes were then incubated overnight at 4 °C with one of the following primary antibodies: mouse anti- Sp Cas9 (Cell Signaling 14697, 1:2000), rabbit anti-TOP3A (Proteintech 14525-1-AP, 1:2000), rabbit anti-RMI1 (Proteintech 14630-1-AP, 1:500), or rabbit anti-ACTB (Proteintech 81115-1-RR) in 5% BSA (VWR AAJ65097) dissolved in TBS-T.

Techniques: Immunoprecipitation, Mass Spectrometry, Flow Cytometry