custom dna string  (Twist Bioscience)

Journal: bioRxiv

Article Title: Mapping the genetic landscape of the DNA damage response with Cas12a-based combinatorial knockout screens

doi: 10.64898/2026.06.07.728858

Figure Lengend Snippet: a , Network representation of top DepMap (24Q4) correlated hits for CIP2A , GEN1, and RHNO1 . The top 4 correlated genes for CIP2A , GEN1, and RHNO1 are shown, with green edges indicating the top correlated gene for the indicated parent node. Displayed numbers are Pearson correlation scores from DepMap. b - h , j , l , n - q , Cells were targeted with the indicated in4mer gRNAs. b , c , Quantification of survival assays and representative survival, respectively, of control (CTRL) and GEN1 KO RPE-1 TP53 -/- cells treated with control (siCTRL) or BRCA1 (siBRCA1) siRNA. Bars represent the mean percentage of survival (± SEM) relative to the CTRL sample treated with siCTRL (n = 3 biological replicates). Statistical significance was determined by unpaired t test. d , Western blot showing BRCA1 and GEN1 levels in RPE-1 TP53 -/- cells examined in survival assays in panels b and c . Tubulin was used as a loading control. e, f , Quantification of survival assays and representative survival, respectively, of CTRL, GEN1, CIP2A, and APEX2 KO RPE-1 TP53 -/- cells treated with siCTRL or siBRCA1. Bars represent the mean percentage of survival (± SEM) relative to the CTRL sample treated with siCTRL (n = 3 technical replicates). Statistical significance was determined by one-way ANOVA. g , Representative image of a metaphase from CTRL RPE-1 TP53 -/- cells quantified in . h , Western blot showing the levels of endogenous (end.) and FLAG-HA-tagged (tag.) GEN1 variants in MCF10A cells examined in survival assays in . The level of endogenous GEN1 is reduced in cells targeted with FANCL-GEN1 in4mer gRNA, while the expression of gRNA-resistant GEN1 constructs is not affected. Tubulin was used as a loading control. i , Western blot confirming expression of Halo-Pol θ in U2OS Halo-Pol θ RHINO-mNeonGreen cells, compared to the parental U2OS cell line (untagged). Total protein levels are shown. j , Western blot confirming expression of RHINO-mNeonGreen in U2OS Halo-Pol θ RHINO-mNeonGreen cells, compared to the parental U2OS cell line (untagged), using an anti-mNeonGreen antibody. RHNO1 was targeted with in4mer gRNA to confirm the specificity of the signal. Asterisk marks a non-specific band. Vinculin was used as a loading control. k , Schematic of the RHNO1 locus showing primer pairs used for PCR (top). Expected size of PCR products is indicated. Genotyping PCR confirming knock-in (KI) of mNeonGreen-tag in the RHNO1 locus of U2OS Halo-Pol θ cells (bottom). Black arrow marks the KI PCR product. l , Western blot showing GEN1 levels in U2OS Halo-Pol θ RHINO-mNeonGreen cells employed in live-cell imaging experiments in and panels n and o . Tubulin was used as a loading control. m Schematic of the protocol used for live-imaging of U2OS Halo-Pol θ RHINO-mNeonGreen cells quantified in (RHINO-mNeonGreen foci) and panels n and o (Halo-Pol θ foci). Asynchronous (async.) cells were synchronized for 16 h in G2/M with 9 µM CDK1 inhibitor (CDK1i; RO-3306) in the presence or absence of 0.4 µM aphidicolin (APH), followed by 10 min labelling of DNA with Hoechst and Halo-Pol θ with JFX65, and subsequent arrest of cells in mitosis with 10 µM S-trityl-L-cysteine (STLC). n , Quantification of mitotic Halo-Pol θ foci in live-cell imaging experiments in U2OS Halo-Pol θ RHINO-mNeonGreen cells. Scatter plot shows individual values (n = 3 biological replicates). At least 24 mitotic cells were examined per sample per experiment. Bars represent the median and interquartile range. Statistical significance was determined by Mann-Whitney test. o , Representative images of mitotic Halo-Pol θ foci in APH-treated cells quantified in panel n . p , q , Quantification of spontaneous CIP2A foci and representative images, respectively, in pre-anaphase mitotic MCF10A cells upon disruption of FANCL and/or GEN1 . Scatter plot shows individual values (n = 3 biological replicates). Bars represent the median and interquartile range. At least 39 pre-anaphase cells were scored per sample per experiment. Statistical significance was determined by Mann-Whitney test. **** = p-value ≤ 0.0001.

Article Snippet: A custom DNA string including the desired modifications was ordered from Twist Biosciences and used to amplify by PCR the fragments before Golden Gate reactions.

Techniques: Control, Western Blot, Expressing, Construct, Knock-In, Live Cell Imaging, Imaging, MANN-WHITNEY, Disruption

Journal: Nature Communications

Article Title: Balancing the efficacy and safety of chimeric antigen receptor T-cell therapy by affinity combination

doi: 10.1038/s41467-026-71354-7

Figure Lengend Snippet: a Schematic of the scFv design for periplasmic expression in E. coli and CAR construct structures for retroviral gene delivery. b Surface plasmon resonance (SPR) analysis of scFv binding kinetics, including dissociation constant ( K D ), association rate ( k on ) and dissociation rate ( k off ). Each dot represents an individual replicate ( n = 3; JCAR021 n = 4, dash indicates the mean measured with the capture of 50 nM scFv for 60 s with a 10 µL/min flow rate). c Schematic representation of epitope mapping to determine similarities in the recognition sites of JCAR017 and JCAR021 scFvs. d SPR sensorgram showing soluble CD19 binding to JCAR021 scFv in the presence or absence of JCAR017 scFv. e Representative flow cytometry plots of transduction efficiency (EGFRt + ) and surface expression level (STII + ) of JCAR017, JCAR021 and reference CAT CAR in primary human T cells. f Quantification of Nur77-tdTomato expression in CAR-transduced Jurkat cells stimulated with CD19 + GFP + Raji tumor cells at an effector-to-target (E:T) ratio of 1:1 or cultured in medium, background-subtracted. Each dot represents the mean of technical triplicates. Bars indicate the mean + SD across biological replicates ( n = 7; CAT n = 5). Additionally, representative histograms displaying Nur77-tdTomato signal are shown. g Intracellular cytokine production quantified after 5 h co-culture with CD19 + GFP + Raji cells at the indicated E:T ratios. Each dot represents the mean of technical triplicates of one independent biological replicate ( n = 3). PMA/ionomycin was used as a positive control. h Representative xCelligence impedance-based killing curves with CD19 + (left) or wildtype (WT) (right) HEK cells, including quantification by the area under the curve (AUC), are shown. Each dot represents a technical replicate of one out of three independent biological experiments. Data is presented as mean + SD ( n = 3). Statistical analyses were performed for ( b , f ) using one-way ANOVA for multiple comparisons between the individual scFvs ( b ) or with JCAR017 as reference ( f ). * p < 0.05, ** p < 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: scFv DNA strings were cloned into a pASG-IBAwt2 vector (IBA Lifesciences) and expressed in electrocompetent E. coli JM83 (IBA Lifesciences).

Techniques: Expressing, Construct, Retroviral, SPR Assay, Binding Assay, Flow Cytometry, Transduction, Cell Culture, Co-Culture Assay, Positive Control

Journal: Nature Communications

Article Title: Balancing the efficacy and safety of chimeric antigen receptor T-cell therapy by affinity combination

doi: 10.1038/s41467-026-71354-7

Figure Lengend Snippet: a Schematic of the scFv sequence used to generate fluorescently labeled scFv FLEXamers along with the CAR sequence for retroviral transduction to construct the affinity library derived from the JCAR021 framework. Mutagenesis regions are indicated by stars. b Principle of the flow cytometry-based assay to assess monomeric scFv:epitope dissociations. c Quantification of scFv dissociation kinetics ( k off -rate) by one-phase exponential decay curve fitting, expressed as dissociation half-life ( t 1/2 ) ( n = 3, 30 min 20 °C). d Comparative analysis of TCR and scFv koff-rates ( n ≥ 3, 30 min 4 °C). e Transduction efficiency (EGFRt + ) (upper left), CAR-expression (STII + ) (upper right) and representative flow cytometry plots (below) in primary human T cells ( n = 3). f Fold change in Nur77-tdTomato expression of CAR-transduced Jurkat cells upon stimulation with CD19 + GFP + Raji cells (E:T 1:1) or incubation in medium for 3 h ( n ≥ 5). Representative flow cytometry plots are shown on the right. g Intracellular cytokine production in CAR-engineered primary human T cells following 5 h co-culture with CD19 + GFP + Raji cells at an E:T of 4:1 ( n ≥ 3) with representative flow cytometry histograms. h Impedance-based xCelligence killing curves (below) and quantification of CAR-T-cell killing (upper) as area under the curve (AUC) ( n ≥ 2). i Heatmap summarizing the functional properties of JCAR021, CAR mfunct and CAR mlowfunct in vitro. f – i Data are normalized to the average value of JCAR017. Data are expressed as mean ± SD. Each dot represents the mean of technical replicates for JCAR017 and JCAR021 and the mean of an independent experiment for the JCAR021 mutants. Statistical analyses were performed in ( e – h ) using a one-way ANOVA test for multiple comparisons with JCAR017 as reference. * p < 0.05, ** p < 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: scFv DNA strings were cloned into a pASG-IBAwt2 vector (IBA Lifesciences) and expressed in electrocompetent E. coli JM83 (IBA Lifesciences).

Techniques: Sequencing, Labeling, Retroviral, Transduction, Construct, Derivative Assay, Mutagenesis, Flow Cytometry, Expressing, Incubation, Co-Culture Assay, Functional Assay, In Vitro