Journal: Advanced Science

Article Title: KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition

doi: 10.1002/advs.202206098

Figure Lengend Snippet: Kmt2d mutation by CRISPR/Cas9 promotes AML in mice. A) Schematic experimental design for mouse modeling using CRISPR/Cas9 system. Trp53 −/− ; Cas9 mouse HSPCs were transduced with mCherry‐linked sg Kmt2d ‐sg Nf1 ‐sg Cas9 or sgScramble‐sg Nf1 ‐sg Cas9 , and then transplanted into sub‐lethally irradiated syngeneic mice. B) Kaplan–Meier survival curves of mice transplanted with Trp53 −/− ; Cas9 HSPCs transduced with sgScramble‐sg Nf1 ‐sg Cas9 (blue; n = 5), sg Kmt2d_ #1‐sg Nf1 ‐sg Cas9 (red, n = 5), or sg Kmt2d_# 2‐sg Nf1 ‐sg Cas9 (orange; n = 5). ** p < 0.01 (log‐rank test). C) WBC, Hb, and PLT counts of sgScramble and sg Kmt2d mice 2 months post‐transplant. Graph represents the mean ± SD; ** p < 0.01, *** p < 0.001, ns, not significant (unpaired two‐tailed t ‐test). D) Representative flow cytometric profiles showing the expression of CD11b/Gr‐1, B220/CD3ε and c‐Kit in bone marrow cells of sacrificed TNKC ( Trp53 −/− ; sg Nf1 ; sg Kmt2d; sg Cas9 ) mice. E) Representative images of histological analyses of blood, spleen, liver, and bone marrow of sacrificed TNKC mice. F) T7 endonuclease I assay on Kmt2d in bone marrow cells of sacrificed TNKC mice. G) Mutation analyses of the Kmt2d regions targeted by CRISPR/Cas9 of TNKC bone marrow cells. Representative Sanger sequences of single TA clones.

Article Snippet: sgRNAs were designed by the ATUM CRISPR gRNA Design tool ( https://www.atum.bio/eCommerce/cas9/input ).

Techniques: Mutagenesis, CRISPR, Transduction, Irradiation, Two Tailed Test, Expressing, T7EI Assay, Clone Assay

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