Journal: bioRxiv

Article Title: Generation and validation of a human iPSC-derived TDP-43 knockout model for ALS disease modeling

doi: 10.64898/2026.04.29.720127

Figure Lengend Snippet: Generation and validation of homozygous TARDBP knockout iPSC clones. (a) CRISPR–Cas9 strategy used to introduce insertion/deletion (INDEL) mutations in exon 1 or exon 2 of TARDBP . (b) Editing efficiency of exon 1– and exon 2–targeting sgRNAs, as determined by ICE (Inference of CRISPR Edits) analysis. (c) qRT–PCR analysis of TARDBP mRNA expression in pooled iPSCs following sgRNA electroporation; One-way ANOVA with Dunnett’s test versus electroporation (EP)-only Control; F (2,9) = 171.0, P < 0.0001; n = 4. (d) Representative immunoblot of TDP-43, STMN2, and α-tubulin in pooled iPSC lysates following TARDBP knockout. (e) Quantification of TDP-43 protein levels in pooled edited iPSCs. Protein abundance was normalized to α-tubulin for each sample and then to EP-only Control; F (4,10) = 51.12, P < 0.0001; n = 3. (f) Quantification of STMN2 protein levels in pooled edited iPSCs. Protein abundance was normalized to α-tubulin for each sample and then to EP-only control cells; F (4,10) = 17.73, P = 0.0002; n = 3. (e, f) One-way ANOVA with Dunnett’s test versus Control. (g) qRT–PCR analysis of full-length TARDBP mRNA expression in individual iPSC clonal lines; F (12,39) = 1260, P < 0.0001; n = 4. (h) qRT–PCR analysis of full-length STMN2 mRNA expression in individual iPSC clonal lines; F (12,39) = 727.6, P < 0.0001; n = 4. (g, h) One-way ANOVA on log-transformed data with Dunnett’s test vs Control 1. Controls 2 and 3 are shown for reference, but statistical significance is annotated only for comparisons with Control 1. (i) RT–PCR analysis of truncated STMN2 cryptic exon ( STMN2 -CE) expression in individual iPSC clonal lines; F (12,39) = 366.4, P < 0.0001; n = 4. One-way ANOVA with Dunnett’s test versus Control 1. (j) Quantification of TDP-43 protein levels in individual iPSC clones using a custom Meso Scale Discovery (MSD) assay; Kruskal–Wallis with Dunn’s test versus Control 1. H = 23.92, P = 0.0208; n = 2. Bars represent mean ± SEM. n de n otes technical replicates per condition. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: sgRNAs targeting exon 1 or exon 2 of TARDBP were designed using Benchling’s CRISPR guide RNA design tool and selected based on predicted high on-target efficiency and low off-target activity.

Techniques: Biomarker Discovery, Knock-Out, Clone Assay, CRISPR, Introduce, Quantitative RT-PCR, Expressing, Electroporation, Control, Western Blot, Quantitative Proteomics, Transformation Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Genome biology

Article Title: SETD2 loss-of-function uniquely sensitizes cells to epigenetic targeting of NSD1-directed H3K36 methylation.

doi: 10.1186/s13059-025-03483-z

Figure Lengend Snippet: Fig. 1 Unbiased genome-wide screening identifies NSD1 as putative SL modifier in SETD2-deficient cells. A Western blot analysis of global H3K36 methylation states in isogenic SETD2-wildtype/mutant HAP1 cells. B Schematic depiction of CRISPR/Cas9 synthetic lethal screen. C Volcano plot highlighting NSD1 as a synthetic lethal hit (SL index: − 1.76; p value = 2.67e − 06). D Gene ontology analysis of the 127 SL candidates identified in the screen reveal enrichment for factors involved in epigenetic remodeling and DNA damage/repair. E Gene-view schematic illustrating inducible deletion of Setd2 in MEFs through Cre-lox excision of exon 6. F PCR genotyping confirming tamoxifen-inducible Cre activity in the Setd2flox/flox parental and Setd2flox/flox; Nsd1−/− MEF cell lines. G Crystal violet staining of Setd2flox/flox and Setd2flox/flox; Nsd1−/− MEF cell lines following treatment with 4-OHT

Article Snippet: Individual sgRNAs for CRISPRi targeting were selected using an in silico CRISPR guide RNA selection tool (Benchling) with corresponding oligos annealed and subcloned by cohesive-end ligation into a lentiviral mU6-(sp)TRACR guide RNA vector following AarI digestion.

Techniques: Genome Wide, Western Blot, Methylation, Mutagenesis, CRISPR, Activity Assay, Staining

Journal: Nature Neuroscience

Article Title: TDP-43-dependent mis-splicing of KCNQ2 triggers intrinsic neuronal hyperexcitability in ALS/FTD

doi: 10.1038/s41593-025-02096-w

Figure Lengend Snippet: a , Schematic of CRISPR–Cas9 editing of embryonic stem cells (ESCs) to generate homozygous KCNQ2 ∆E5/∆E5 lines and differentiation into cortical excitatory neurons. b , RT–PCR shows that KCNQ2 ∆E5/∆E5 neurons express KCNQ2 ∆E5 and KCNQ2 WT/WT express KCNQ2 WT . c , Representative immunocytochemical images of neurons stained with DAPI, MAP2, KCNQ2 and ANK-G. Arrowheads denote the beginning of AIS. Asterisk (*) denotes KCNQ2 localization in the AIS for WT (top) and accumulated in the soma for KCNQ2 ∆E5/∆E5 neurons (bottom). Yellow dashed line outlines the cell body. Scale bar, 10 μm. d , Percentage of WT ( n = 51, 0%) and KCNQ2 ∆E5/∆E5 ( n = 79, 100%) neurons with somatic accumulation of KCNQ2. e , Quantification of KCNQ2 signal intensity variation. Statistical significance determined by unpaired, two-tailed Student’s t -test. Data are shown as mean ± s.e.m.; each circle corresponds to one neuron. a.u., arbitrary units. f , Representative image of KCNQ2 ∆E5/∆E5 neuron stained with DAPI, MAP2, calnexin and KCNQ2. Top, maximum Z -projection; middle, 3D views with neuron rotated forward; bottom, 3D views from below. g , Representative raster plot of neuronal activity recorded in a MEA well for control (top) and KCNQ2 ∆E5/∆E5 (bottom). Rows depict individual electrodes; black lines represent single spikes; blue lines indicate ‘bursts’. h – o , Longitudinal analysis of neuronal MEA recordings for days 9–43 ( h – k ) or 12–43 ( l – o ). Data are presented as means from n = 3 independent experiments ( n = 59 wells for WT and n = 64 for KCNQ2 ∆E5/∆E5 ); circles represent means; shaded areas, s.e.m. Two-way repeated-measures ANOVA was used for h , i and l ; mixed-effects model restricted maximum likelihood) for j , k , m , n and o . P values: in black indicate genotype effects and in pink reflect genotype × day interactions. MEA metrics are indicated within each panel. p , Experimental schematic (top) and representative raster plot from MEA wells (bottom) during treatment with the K v 7 agonist ICA-069673 (1 μM). For each metric, pre-ICA-069673 and post-ICA-069673 values are represented as the percent of baseline values (right). Each circle-pair represents the change in activity recorded from a well (total number of wells from two replicate MEA plates were combined for analysis: n = 20 for WT and n = 19 wells for KCNQ2 ∆E5/∆E5 . P value determined by unpaired, two-tailed Student’s t -test.

Article Snippet: Four guide RNAs (gRNAs) targeting introns 4 and 5 of KCNQ2 (Extended Data Fig. ) were designed using the CRISPR wizard with default settings on Benchling (2021, https://benchling.com ). gRNA oligonucleotides were purchased from IDT with BbsI sticky ends to clone into an expression vector driven by the human U6 promoter (custom synthesis, Broad Institute).

Techniques: CRISPR, Reverse Transcription Polymerase Chain Reaction, Staining, Two Tailed Test, Activity Assay, Control

Journal: Nature Neuroscience

Article Title: TDP-43-dependent mis-splicing of KCNQ2 triggers intrinsic neuronal hyperexcitability in ALS/FTD

doi: 10.1038/s41593-025-02096-w

Figure Lengend Snippet: ( a ) Details of CRISPR mutagenesis of KCNQ2 . The four gRNAs targeting KCNQ2 are presented along with details of deletions induced in and KCNQ2 ∆E5/∆E5 cells. ( b ) KCNQ2 allele copy number assay for unrelated iPSC cell line, isogenic control and KCNQ2 ∆E5/∆E5 ESCs. (c) Karyotype results for isogenic control and KCNQ2 ∆E5/∆E5 ESCs. ( d ) Representative images of NGN2 cortical neurons stained with DAPI, GFP, and MAP2. ( e ) Representative images of NGN2 cortical neurons stained with DAPI, MAP2 and KCNQ2. Scale bar: 25 μm. Yellow dashed line outlines neuronal cell body. Letters signify individual neurons for which greyscale images of MAP2 and KCNQ2 signal are magnified. Scale bar: 10 μm. (f) Quantification of the mean KCNQ2 signal was significantly higher in KCNQ2 ∆E5/∆E5 neurons (p < 0.0001). ( g ) Quantification of the max KCNQ2 signal was also significantly higher in KCNQ2 ∆E5/∆E5 neurons (p = 0.0035). Statistical significance for (f-g) was determined by unpaired, two-tailed student’s t-test. Data are shown as mean ± SEM, each circle corresponds to one neuron (control: n = 51, KCNQ2 ∆E5/∆E5 : n = 79).

Article Snippet: Four guide RNAs (gRNAs) targeting introns 4 and 5 of KCNQ2 (Extended Data Fig. ) were designed using the CRISPR wizard with default settings on Benchling (2021, https://benchling.com ). gRNA oligonucleotides were purchased from IDT with BbsI sticky ends to clone into an expression vector driven by the human U6 promoter (custom synthesis, Broad Institute).

Techniques: CRISPR, Mutagenesis, Control, Staining, Two Tailed Test