px602 vector  (Addgene inc)

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: In vivo genome editing of the tyrosine catabolic pathway by rAAV8-SaCas9 in C57BL/6N mice. a) The tyrosine degradation pathway and associated inborn errors of metabolism (IEMs). The catabolic reaction inhibited by NTBC is indicated in red. 4-HPP, 4-hydroxyphenylpyruvate; HGA, homogentisic acid; MAA, maleylacetoacetate; FAA, fumarylacetoacetate. b) Top: Schematic of the rAAV-SaCas9 vector. The thyroxine-binding globulin (TBG), bovine growth hormone polyadenylation (bGHpA), and hU6 promoter sequences are indicated. Bottom: Sequences of the sgRNAs targeting Hgd, Hgd, and Gstz1 chosen for in vivo studies. The protospacer adjacent motifs (PAMs) are annotated. The two blue nucleotides in the Gstz1 target site are mismatches compared to the human sequence. c) Neonatal male C57BL/6N pups were injected with 5 × 10 10 vector genomes (VGs) of rAAV8-SaCas9 into the retro-orbital sinus, weaned at 21 days old, and sacrificed at 40 days of age. Mice were assayed for phenotypic and metabolic modifications after weaning. Kaplan-Meier survival curves of male C57BL/6N mice injected at 2 days old with 5 × 10 10 VGs rAAV8-SaCas9 or saline into the retro-orbital sinus. The numbers of mice per group ( n ) and rAAV8 targets are indicated. d) Glycemia of non-fasted mice. Solid lines indicate the mean and the shaded areas denote the SEM. e) Urine succinylacetone and f) homogentisic acid levels in mice treated as in c) were determined 35 days after weaning. Samples were collected over 24 hours using metabolic cages containing 2–3 mice . Also indicated for each group of animals are the mean and the standard error of the mean (SEM). The detection limits for succinylacetone and homogentisic acid were 0.1 mmol/mol and approximately 3 mmol/mol creatinine, respectively. BDL: below detection limit. g) Genomic whole-liver and kidney DNA was extracted, and Surveyor assays were used to determine indel frequencies. Each symbol represents a different animal. Also indicated for each group of animals are the mean and the standard error of the mean (SEM). A mouse injected with saline was used as the negative control for the Surveyor assay.

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 ( Ran et al . 2015 ) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: In Vivo, Plasmid Preparation, Binding Assay, Sequencing, Injection, Saline, Negative Control

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: Metabolic rewiring through liver-specific knockout of Hpd, Hgd, and Gstz1 in Fah −/− mice. a) Experimental design for in vivo editing. Neonatal (2-day-old) male Fah −/− pups were injected with 5 × 10 10 VGs of rAAV8-SaCas9 or saline into the retro-orbital sinus and weaned at 21 days old. NTBC treatment was stopped at different time points; 28 days (saline, n = 3; Hpd, n = 7; Hgd , n = 3; Gstz1 , n = 3), 30 days ( Hpd, n = 6; Gstz1 , n = 3), 41 days (saline, n = 2; Gstz1 , n = 7), or 44 days ( Hgd , n = 7). Since the period elapsed between weaning and NTBC removal did not affect the outcome, mice from each treatment group were combined in the graphs shown in (b-d). The numbers of mice per group ( n ) and rAAV targets are indicated. b) Kaplan-Meier survival curves following NTBC removal. Mice were sacrificed after losing 20% of their body weight. All animals injected with rAAV8-SaCas9 experienced statistically significant differences in survival as determined per the log-rank test when compared to a group of saline-injected animals, whose median survival was 21 days: Hpd -treated animals, complete survival ( P < 0.0001); Hgd -treated animals, median survival of 4 days ( P < 0.001), Gstz1 -treated animals, median survival of 5 days ( P < 0.001). c) Body weight was measured daily following NTBC removal. Solid lines indicate the mean and shaded areas denote the standard error of the mean (SEM). d) Glycemia was monitored in non-fasted mice. e) Urine succinylacetone and f) homogentisic acid levels were determined 24 hours before NTBC removal and again 24 hours before the predicted time of sacrifice (1, 4, and 19 days post-NTBC removal for mice treated with Hgd rAAV8, Gstz1 rAAV8, and saline, respectively. Levels were determined in Hpd rAAV8-treated mice 34 days after NTBC removal. Samples were collected over 24 hours using metabolic cages containing 2–5 mice . Also indicated for each group of animals are the mean and the standard error of the mean (SEM). Statistical analysis was performed with 2-way ANOVA followed by Tukey tests for the represented pairwise comparisons. * : P < 0.05; ** : P < 0.01; **** : P < 0.0001. The detection limits for succinylacetone and homogentisic acid were 0.1 mmol/mol and approximately 3 mmol/mol creatinine, respectively. BDL: below detection limit. g) Genomic DNA was extracted from whole livers of mice treated with Hpd -, Hgd- and Gstz1- targeting vectors sacrificed either at 30 days of age with continuous NTBC treatment or at time of sacrifice after NTBC removal (respectively one year for Hpd -targeted animals, 3–4 days for Hgd -targeted animals and 4–6 days for Gstz1 -targeted animals), and Surveyor assays were used to determine indel frequencies. Each symbol represents a different animal. Also indicated for each group of animals are the mean and the standard error of the mean (SEM). A mouse injected with saline was used as the negative control for the Surveyor assay.

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 ( Ran et al . 2015 ) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: Knock-Out, In Vivo, Injection, Saline, Negative Control

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: Tissue analysis of Fah−/− mice following metabolic rewiring by rAAV8-SaCas9. a) Kidney and liver sections from Fah−/− mice treated with saline or rAAV8-SaCas9 vectors targeting Hpd, Hgd, or Gstz1 as in . Fah −/− mice injected with saline and the Hpd, Hgd, and Gstz1 rAAV8 s were sacrificed 20, 35, 2, and 5 days after NTBC removal, respectively. Top panel: Representative Masson's trichrome-stained kidney sections from the treatment groups. Magnification: 10×. Bottom panel: Representative hematoxylin and eosin-stained liver sections from the treatment groups. PV: portal vein; black arrow: portal and lobular inflammation; dashed arrow: ductular proliferation; white arrow: necrosis; red arrow: ballooning degeneration. Magnification: 200×. b) Representative kidneys from Fah −/− mice that were treated with saline and kept on NTBC (left) or treated with a vector targeting Hgd with NTBC withdrawn (right). c) Urine samples (1 µl per well) from treated Fah −/− animals on (+) and off (−) NTBC and untreated controls (as described in ) were loaded on a 4–15% gradient mini-PROTEAN TGX Stain-Free gel before electrophoresis, Coomassie staining, and imaging. Urine from a C57BL/6N male mouse was used as a negative control. A band of the size expected for mouse serum albumin is indicated with an arrow. Also indicated with an arrow are bands of the expected size for the mouse major urinary proteins (MUPs).

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 ( Ran et al . 2015 ) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: Saline, Injection, Staining, Plasmid Preparation, Electrophoresis, Imaging, Negative Control

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: Metabolic rewiring by inactivation of Hgd or Gstz1 yields similar effects in both female and male Fah−/− mice. a) Neonatal pups of both sexes were injected at birth with 5 × 1010 VGs of rAAV8-SaCas9 and NTBC was withdrawn at 28 days of age. Mice were sacrificed after losing 20% of their body weight. Kaplan-Meier survival curves of female (f; solid lines) and male (m; dotted lines) Fah−/− animals following NTBC withdrawal. b) Body weight was measured daily following NTBC removal. Solid or dotted lines indicate the mean and shaded areas denote the standard error of the mean (SEM). c) Glycemia was monitored in non-fasted mice. d) Representative kidneys from female Fah−/− mice of a similar age that were either untreated and kept on NTBC (left) or treated with a vector targeting Hgd (right) after NTBC withdrawal. e) Genomic DNA was extracted from whole livers of mice treated with Hgd- and Gstz1-targeting vectors meeting the sacrifice endpoint, and Surveyor assays were used to determine indel frequencies. Each symbol represents a different animal. Female animals are represented as triangles, whereas males are represented as circles. Also indicated for each group of animals are the mean and the standard error of the mean (SEM). A mouse injected with saline was used as the negative control for the Surveyor assay.

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 ( Ran et al . 2015 ) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: Injection, Plasmid Preparation, Saline, Negative Control

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: Model of metabolic rewiring in Fah−/− mice following liver-specific knockout of tyrosine catabolic enzymes. Observed disease phenotypes and survival outcomes after injecting neonatal Fah−/− pups with saline or liver-specific rAAV8-SaCas9 vectors targeting Hpd, Hgd, or Gstz1 and NTBC withdrawal. The main metabolites predicted to be produced by the liver and unedited kidneys in each condition are shown. Fumarylacetoacetate (FAA) and maleylacetoacetate (MAA) accumulation leads to non-enzymatic production of succinylacetone (SA) (see ).

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 ( Ran et al . 2015 ) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: Knock-Out, Saline, Produced

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1.

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: Fig. 3. Tissue analysis of Fah−/− mice following metabolic rewiring by rAAV8-SaCas9. a) Kidney and liver sections from Fah−/− mice treated with saline or rAAV8-SaCas9 vectors targeting Hpd, Hgd, orGstz1 as in Fig. 2. Fah−/− mice injected with saline and the Hpd, Hgd, and Gstz1 rAAV8 s were sacrificed 20, 35, 2, and 5 days after NTBC removal, respectively. Top panel: Representative Masson’s trichrome-stained kidney sections from the treatment groups. Magnification: 10×. Bottom panel: Representative hematoxylin and eosin-stained liver sections from the treatment groups. PV: portal vein; black arrow: portal and lobular inflammation; dashed arrow: ductular proliferation; white arrow: necrosis; red arrow: ballooning degeneration. Magnification: 200×. b) Representative kidneys from Fah−/− mice that were treated with saline and kept on NTBC (left) or treated with a vector targeting Hgd with NTBC withdrawn (right). c) Urine samples (1 µl per well) from treated Fah−/− animals on (+) and off (−) NTBC and untreated controls (as described in Fig. 2) were loaded on a 4–15% gradient mini-PROTEAN TGX Stain-Free gel before electrophoresis, Coomassie staining, and imaging. Urine from a C57BL/6N male mouse was used as a negative control. A band of the size expected for mouse serum albumin is indicated with an arrow. Also indicated with an arrow are bands of the expected size for the mouse major urinary proteins (MUPs).

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 (Ran et al. 2015) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: Saline, Injection, Staining, Plasmid Preparation, Electrophoresis, Imaging, Negative Control

Journal: Genetics

Article Title: In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1.

doi: 10.1093/genetics/iyae139

Figure Lengend Snippet: Fig. 4. Metabolic rewiring by inactivation of Hgd or Gstz1 yields similar effects in both female and male Fah−/− mice. a) Neonatal pups of both sexes were injected at birth with 5 × 1010 VGs of rAAV8-SaCas9 and NTBC was withdrawn at 28 days of age. Mice were sacrificed after losing 20% of their body weight. Kaplan-Meier survival curves of female (f; solid lines) and male (m; dotted lines) Fah−/− animals following NTBC withdrawal. b) Body weight was measured daily following NTBC removal. Solid or dotted lines indicate the mean and shaded areas denote the standard error of the mean (SEM). c) Glycemia was monitored in non-fasted mice. d) Representative kidneys from female Fah−/− mice of a similar age that were either untreated and kept on NTBC (left) or treated with a vector targeting Hgd (right) after NTBC withdrawal. e) Genomic DNA was extracted from whole livers of mice treated with Hgd- and Gstz1-targeting vectors meeting the sacrifice endpoint, and Surveyor assays were used to determine indel frequencies. Each symbol represents a different animal. Female animals are represented as triangles, whereas males are represented as circles. Also indicated for each group of animals are the mean and the standard error of the mean (SEM). A mouse injected with saline was used as the negative control for the Surveyor assay.

Article Snippet: Following in vitro screening, selected SaCas9 sgRNAs were cloned into the thyroxine-binding globulin (TBG)-driven SaCas9 nuclease rAAV vector pX602 (Ran et al. 2015) (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.

Techniques: Injection, Plasmid Preparation, Saline, Negative Control