crispr wizard  (Benchling Inc)

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