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

Journal: Nature

Article Title: In Vivo Base Editing Rescues Hutchinson-Gilford Progeria Syndrome in Mice

doi: 10.1038/s41586-020-03086-7

Figure Lengend Snippet: ( a ) Bystander Val690Ala editing in progeria patient-derived HGADFN167 and HGADFN188 cells 20 days after treatment with lentiviral ABE7.10max-VRQR. ( b ) Indel formation frequency at the c.1824 target locus in HGADFN167 and HGADFN188 cells 20 days after treatment with lentiviral ABE7.10max-VRQR. Values and error bars represent mean±SD for n=5 technical replicates (individual points) for (a) and (b). ( c ) Sanger DNA sequencing traces of untreated HGADFN167 cells, 20-day treated HGADFN167 cells, and unaffected control cells. The target nucleotide is boxed. ( d ) qPCR-normalized progerin mRNA abundance in cells described in c . Values and error bars represent mean±SD for n=3 biological replicates. ( e ) Western blot analysis of HGADFN167 cells described in c . LMNA, progerin, and LMNC protein are all stained on the gel, A GAPDH loading control is shown below. An additional replicate is provided in . Unaff. ctrl, control cells from an unaffected parent. ( f ) Sanger DNA sequencing traces of untreated, non-targeting (NT) sgRNA treated, and ABE treated HGADFN155 fibroblasts at 20d and 30d time-points to ensure NT-sgRNA did not lead to DNA editing. ( g ) Western blot analysis of cells described in f. LMNA, progerin, and LMNC proteins are all stained on the gel, A β-actin loading control is shown below. Expected molecular weights: lamin A, 74 kDa; progerin, 69 kDa; lamin C, 65 kDa. Complete blots are available in . Additional replication was not performed. ( h ) HGADFN167 (left) and HGADFN188 (right) cell lines untreated or treated with lentiviral ABE7.10max-VRQR after 10 or 20 days show similar relative distributions of A-to-I single-nucleotide variants (SNVs) in their transcriptomes compared with the hg38 human genome reference sequence. On average, 36±3.6% of SNPs in these samples occur with ~100% frequency, suggesting they arise from genomic sequence variations; however, we cannot explicitly exclude them from consideration since no whole genome sequence is available for these cell lines. Raw counts of 100% edited SNPs per sample are: Untreated HGADFN167 cells (849), HGADFN167 10 d after treatment (883), HGADFN167 20 d after treatment (871), Untreated HGADFN188 cells (488), HGADFN188 10 d after treatment (501), HGADFN188 20 d after treatment (510).

Article Snippet: Cas9-VRQR was complexed with a synthetic sgRNA (Synthego) containing phosphorothioate linkage and 2’MeO modification at the first and last three nucleotides, and this complex was used to treat circularized DNA.

Techniques: Derivative Assay, DNA Sequencing, Control, Western Blot, Staining, Sequencing

Journal: Nature

Article Title: In Vivo Base Editing Rescues Hutchinson-Gilford Progeria Syndrome in Mice

doi: 10.1038/s41586-020-03086-7

Figure Lengend Snippet: ( a ) Dual AAV9 encoding split-intein ABE7.10max-VRQR base editor halves and the LMNA -targeting sgRNA were injected into progeria mice. P3 retro-orbital (RO) injections (5×10 10 of each AAV vg, 1×10 11 vg total), P14 RO injections (5×10 11 vg of each AAV vg, 1×10 12 vg total), or P14 intraperitoneal (IP) injections (5×10 11 vg of each AAV, 1×10 12 vg total) were administered. ( b ) DNA editing efficiencies correcting LMNA c.1824 from T (pathogenic) to C (wild-type) for P3 RO-injected mice (left) or P14 RO-injected mice (right) in 6-week- or 6-month-old mice. Editing in P14 IP-injected mice is in . ( c ) ddPCR counts for human LMNA (grey bars) and progerin (red bars) RNA transcript abundance in P14 RO saline- or ABE-AAV9-injected mice in liver and heart. See for additional data. ( d ) Western blot analysis of human lamin A, progerin, and lamin C proteins in liver and heart of P14 RO saline- or ABE-AAV9-injected mice. Each lane shows tissue from a different mouse. WT is a C57BL/6 mouse lacking the transgene, showing that the antibody is specific to human lamin proteins. See for additional data. Values and error bars represent mean±SD for the indicated number of biological replicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Student’s unpaired two-sided t-test.

Article Snippet: Cas9-VRQR was complexed with a synthetic sgRNA (Synthego) containing phosphorothioate linkage and 2’MeO modification at the first and last three nucleotides, and this complex was used to treat circularized DNA.

Techniques: Injection, Saline, Western Blot

Journal: Nature

Article Title: In Vivo Base Editing Rescues Hutchinson-Gilford Progeria Syndrome in Mice

doi: 10.1038/s41586-020-03086-7

Figure Lengend Snippet: ( a ) Dual AAV9 encoding split-intein ABE7.10max-VRQR base editor halves and the LMNA -targeting sgRNA were injected into homozygous human LMNA c.1824 C>T mice. P3 retro-orbital (RO) injections (5×10 10 of each AAV vg, 1×10 11 vg total), P14 RO injections (5×10 11 of each AAV vg, 1×10 12 vg total), and P14 intraperitoneal (IP) injections (5×10 11 of each AAV vg, 1×10 12 vg total) were tested. At 6-weeks of age, mice were harvested and heart, muscle, liver, aorta, and bone were isolated for sequencing analysis. Tissues were sub-sectioned for sequencing analysis to ensure sub-sections did not show differences in editing efficiencies for downstream analyses. Each bar represents a different tissue subsection. DNA editing efficiencies correcting LMNA c.1824 from T (pathogenic) to C (wild-type) for P3 RO-injected mice (left, n=4), P14 RO-injected mice (middle, n=5), and P14 RO-injected mice (right, n=5) at 6 weeks of age are shown for five disease-relevant tissues. Values and error bars represent mean±SD. ( b ) Apparent LMNA c.1824 T (pathogenic) to C (wild-type) mutations from tissue samples of saline-injected P3 RO (left) and P14 RO (right) control mice at 6 months of age show background signal due to amplicon crossover during PCR between the human diseased allele and the wild-type mouse allele, which share 90% overall sequence identity within the amplified region. Similar crossover levels were observed across 11 tissues in both P3 RO and P14 RO saline-injected mice. Values and error bars represent mean±SD for n=12 mice (6 male, 6 female). ( c ) Computational filtering of same sequencing reads shown in (b) after removing any reads containing any mouse-specific sequence variations, analyzing only reads containing exclusively human sequence. The script used to remove mouse-containing sequencing reads is in and is described in the methods. WAT white adipose tissue. ( d ) DNA editing for P3- and P14-injected mice at 6 months of age across 11 tissues. Each point represents a biological replicate of a tissue harvested from a unique mouse (n=12 for each group). ( e ) Val690➔Ala bystander editing frequency across eleven tissues for P3 RO and P14 RO ABE-treated mice at 6 months of age (n=12 for each group). ( f ) Indel frequencies at the c.1824 target locus across 11 tissues for P3 RO and P14 RO ABE-treated mice at 6 months of age. Values and error bars represent mean±SD for the indicated number of biological replicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Student’s unpaired two-sided t-test.

Article Snippet: Cas9-VRQR was complexed with a synthetic sgRNA (Synthego) containing phosphorothioate linkage and 2’MeO modification at the first and last three nucleotides, and this complex was used to treat circularized DNA.

Techniques: Injection, Isolation, Sequencing, Saline, Control, Amplification