Journal: Molecular Therapy. Nucleic Acids

Article Title: A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases

doi: 10.1016/j.omtn.2024.102130

Figure Lengend Snippet: hADAR2- and hADAR1-mediated editing of selected IRD mutations can be induced in the presence of gRNAs (A) Schematic representation of the dsRNA structure formed when the “tail” containing the selected gRNA sequences folds back at the RNA level on the target to form the dsRNA structure for ADAR recruitment. (B) A table describing the contents of the strains (denoted as I–V) tested in (C)–(F). Strain I mimicked the outcome of perfect editing by ADAR and served as the positive control, while the negative controls (strains IV and V) carried an “hADAR1-mediated editing of selected empty” LEU2 plasmid without the ADAR enzyme. (C and D) Growth curves of the indicated strains were produced as described in two dimensions in the presence of hADAR2 (top) and hADAR1 (bottom). The numbers representing the editing levels at the target A’s are the average (n = 3) obtained on NGS analysis.

Article Snippet: All chemically modified gRNAs used for the editing of IRD mutations were purchased as high-performance liquid chromatography (HPLC)-cleaned, 60-mer RNAs at a 4–6 nmol scale from Biospring.

Techniques: Positive Control, Plasmid Preparation, Produced

Journal: STAR Protocols

Article Title: Protocol for generation of humanized HCC mouse model and cancer-driver mutations using CRISPR-Cas9

doi: 10.1016/j.xpro.2023.102389

Figure Lengend Snippet: Tumor progression and poor survival rate in HCC patients with high expression of NANOG and EZH2 (A) CRISPR-Cas9-based gene editing generated β-Catenin constitutively-active alanine substitutions as indicated (Ser33, Ser37, Thr41, Ser45). ARID1A and CTNNB1 (β-catenin) mutations by sgRNA and donor DNA. Distribution of the mutations detected by Exome-seq in the coding region of ARID1A gene is shown in cancer from a literature. (B) Humanized HCC Mouse Model. The diagram depicts the humanized HCC model production procedure (1) transplantation of human fetal liver cells into FRG neonates; (2) NTBC withdrawal; (3) Ad:uPA injection; (4) X-ray irradiation; (5) hepatoblast gene editing; (6) Intrahepatic injection of both LPCs with non-parenchymal cells; (7) Withdrawal metabolic rescue drug NTBC from drinking water. Humanized FRG -hu-Hep/HSC were fed alcohol Western diet (WD) for tumor development. Days refer to the procedure timeline. (C) Immunoblot of ARID1A of livers collected from mice shown in (D). (D) Alcohol Western diet (WD) feeding promoted HCC in humanized FRG -hu-Hep/HSC mice. Tumor incidence in mice, effects of alcohol and western diet (WD) as indicated. Liver tumor incidence rate markedly increases by ARID1A/CTNNB1 mutations and alcohol Western alcohol diet feeding. Part of Figure is being published after acceptance in Cell Death Discovery article (1) and another pending manuscript.

Article Snippet: Target-specific chemically modified sgRNA: Synthetic sgRNA Kit , Synthego , cat. no. A29377.

Techniques: Expressing, CRISPR, Generated, Transplantation Assay, Injection, Irradiation, Western Blot

Journal: STAR Protocols

Article Title: Protocol for generation of humanized HCC mouse model and cancer-driver mutations using CRISPR-Cas9

doi: 10.1016/j.xpro.2023.102389

Figure Lengend Snippet:

Article Snippet: Target-specific chemically modified sgRNA: Synthetic sgRNA Kit , Synthego , cat. no. A29377.

Techniques: Staining, Virus, Control, Infection, Western Blot, Modification, Purification, Recombinant, Red Blood Cell Lysis, Lysis, Enzyme-linked Immunosorbent Assay, Isolation, Positive Control, Transfection, CRISPR, Sterility, Gene Knock-In, Sequencing, Software, Flow Cytometry, Irradiation