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: Blood Advances

Article Title: CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

doi: 10.1182/bloodadvances.2020004112

Figure Lengend Snippet: Efficient CRISPR/Cas9 deletions of platelet function genes are maintained in CD34+-derived MKs. (A) Overview of strategy. CD34+ cells isolated from cord blood were transfected on day 5 (D5) (unless otherwise noted) with RNP complexes containing tracrRNA, gene targeting crRNA, and Cas9 protein. MK assays, including assays normally used to measure platelet (PLT) functional responses, were performed on day 13 of culture. (B) CD34+ cells were transfected on different days of culture with negative (Neg) control or ITGA2B crRNA. Surface expression of αIIb (% CD41+ cells; see Figure 2B for gating) was measured by flow cytometry on day 13 MKs. (C-F) Western blot and densitometry analysis of proteins on day 13 MKs after targeting by negative control or the indicated gene specific CRISPR on day 5. Data are presented as mean ± standard error of the mean (SEM) (3 independent cords per group). Unpaired Student t test: *P < .05; **P < .01; ***P < .001. CalDAG, calcium diacylglycerol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: All materials and reagents were kept sterile and RNAse free throughout. crRNA, trans-activating CRISPR RNA (tracrRNA) (IDT #1072532), and electroporation enhancer (IDT #1075915) were resuspended to 100 µM in 10 mM tris(hydroxymethyl)aminomethane (Tris; pH 7.5), and stored at −20°C until use. crRNA and tracRNA were mixed 1:1 (2.5 µL each) in a polymerase chain reaction (PCR) tube and heated at 95°C for 5 minutes in a thermal cycler with a heated lid, followed by cooling to room temperature on a bench top for at least 10 minutes before storing at −20°C.

Techniques: CRISPR, Derivative Assay, Isolation, Transfection, Functional Assay, Expressing, Flow Cytometry, Western Blot, Negative Control

Journal: Blood Advances

Article Title: CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

doi: 10.1182/bloodadvances.2020004112

Figure Lengend Snippet: Effect of COMMD7 KO on megakaryopoiesis. (A) Real-time PCR analysis of COMMD7 RNA expression in COMMD7 CRISPR-treated day 13 MKs. Data are normalized to GAPDH (3 independent cords per group). (B) Western blot of COMMD7 protein in platelets from 3 healthy donors and in control and COMMD7 KO MKs from 3 independent cords per group. β-actin (ACTB) was used as loading control. (C) Percentage of viable negative control or COMMD7 KO cells on day 13 of culture. (D) Proplatelet formation on day 13 MKs plated overnight on fibrinogen and stained with Phalloidin 488. Shown are representative images of 10 random images taken from each of 3 cords. The percentage of control or COMMD7 KO MKs making proplatelets on day 13 was counted blinded to group and summarized in the bar graph on the right. (E) Flow cytometry analysis of MK maturation markers on day 13 control or COMMD7 MKs. Viable MKs were gated as in Figure 2B. Shown is the mean ± SEM of the percent of cells (y-axis) expressing MK maturation markers. Student t test: *P < .05; **P < .01.

Article Snippet: All materials and reagents were kept sterile and RNAse free throughout. crRNA, trans-activating CRISPR RNA (tracrRNA) (IDT #1072532), and electroporation enhancer (IDT #1075915) were resuspended to 100 µM in 10 mM tris(hydroxymethyl)aminomethane (Tris; pH 7.5), and stored at −20°C until use. crRNA and tracRNA were mixed 1:1 (2.5 µL each) in a polymerase chain reaction (PCR) tube and heated at 95°C for 5 minutes in a thermal cycler with a heated lid, followed by cooling to room temperature on a bench top for at least 10 minutes before storing at −20°C.

Techniques: Real-time Polymerase Chain Reaction, RNA Expression, CRISPR, Western Blot, Negative Control, Staining, Flow Cytometry, Expressing

Journal: Life

Article Title: CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

doi: 10.3390/life11010013

Figure Lengend Snippet: The mechanism of action of clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9). The guide RNA (gRNA) molecule directs Cas9 protein to the target DNA and Cas9 cleaves genomic DNA 3–4 bp upstream of the PAM (protospacer-adjacent motif) site.

Article Snippet: Some available webtools include, CRISPy ( www.crispy.secondarymetabolites.org ) [ ], CRISPR-ERA ( www.crispr-era.stanford.edu ) [ ], E-CRISPR ( www.e-crisp.org ) [ ], Benchling ( www.benchling.com ), or ATUM gRNA design tool ( www.atum.bio ).

Techniques: CRISPR

Journal: Life

Article Title: CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

doi: 10.3390/life11010013

Figure Lengend Snippet: Some CRISPR-based applications in Saccharomyces cerevisiae . ARS: autonomously replicating sequence; CEN: yeast centromere.

Article Snippet: Some available webtools include, CRISPy ( www.crispy.secondarymetabolites.org ) [ ], CRISPR-ERA ( www.crispr-era.stanford.edu ) [ ], E-CRISPR ( www.e-crisp.org ) [ ], Benchling ( www.benchling.com ), or ATUM gRNA design tool ( www.atum.bio ).

Techniques: CRISPR, Sequencing, Expressing, Transformation Assay, Plasmid Preparation, Activation Assay