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: Circulation research

Article Title: In Vivo Ryr 2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia

doi: 10.1161/CIRCRESAHA.118.313369

Figure Lengend Snippet: A, Schematic of gRNA design to target R176Q mutant Ryr2 allele in genomic DNA of R176Q/+ mouse. The mutant codon resulting in the R176Q mutation (red) and the silent Rsr restriction site (green) are marked in the sequence. The guide sequence (22-nt, blue) pairs with the DNA target marked as blue bar upstream of a 5’-NNGRRT adjacent motif (PAM; orange). Cas9 will mediate a double-stranded break ~3 bp upstream of the PAM (red triangle). B, Schematic representation of the SaCas9 AAV9 constructs. Original backbone with BbsI cloning sites is used as control.

Article Snippet: The AAV-CRISPR vector, 1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA was generated by the Lagor lab (Addgene plasmid 109320) based on the SaCas9 transgene from px602, Addgene plasmid 61593 (gift of Dr. Feng Zhang).

Techniques: Mutagenesis, Sequencing, Construct, Cloning, Control

Journal: Circulation research

Article Title: In Vivo Ryr 2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia

doi: 10.1161/CIRCRESAHA.118.313369

Figure Lengend Snippet: A, Experimental timeline of AAV9 injections and subsequent phenotypical analysis B, Representative examples of simultaneous recording of surface ECG lead I (L-1) and intracardiac ventricular electrogram showing sustained polymorphic ventricular tachycardia in a R176Q/+ mouse treated with placebo (AAV9-Bbsl-SaCas9; left) and normal sinus rhythm in an AAV9-gRNA-SaCas9 treated R176Q/+ mouse (right). C, Bar graph summarizing the incidence of inducible sustained VT in R176Q/+ mice and WT littermates treated with AAV9-Bbsl-SaCas9 or AAV9-gRNA-SaCas9, respectively. Abbreviations: P, postnatal day; SC, subcutaneous. *p<0.05.

Article Snippet: The AAV-CRISPR vector, 1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA was generated by the Lagor lab (Addgene plasmid 109320) based on the SaCas9 transgene from px602, Addgene plasmid 61593 (gift of Dr. Feng Zhang).

Techniques:

Journal: Circulation research

Article Title: In Vivo Ryr 2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia

doi: 10.1161/CIRCRESAHA.118.313369

Figure Lengend Snippet: A, Diagram showing where PCR primers bind to sequences in exons 3-6 upstream of mutation R176Q in exon 8 (green) and primers targeting exons 11-13 downstream of R176Q (orange). B, Levels of RyR2 mRNA in cardiac tissue from R176Q/+ mice or WT littermates, treated with BbsI-SaCas9 or gRNA-SaCas9. C, Western blot analysis of RyR2 and JPH2 protein levels in heart tissue from R176Q/+ mice or WT littermates. D, Corresponding quantifications of RyR2, and E, JPH2 proteins levels normalized to GAPDH. *p<0.05.

Article Snippet: The AAV-CRISPR vector, 1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA was generated by the Lagor lab (Addgene plasmid 109320) based on the SaCas9 transgene from px602, Addgene plasmid 61593 (gift of Dr. Feng Zhang).

Techniques: Mutagenesis, Western Blot

Journal: Circulation research

Article Title: In Vivo Ryr 2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia

doi: 10.1161/CIRCRESAHA.118.313369

Figure Lengend Snippet: A, Schematic picture showing gRNA-SaCas9 and “reporter” AAV9 co-injected in R176Q/+ mice. The “reporter” virus with cloned targeted sequence was cut by gRNA-SaCas9 but not the control BbsI-SaCas9. The mCherry protein is only expressed in cardiomycytes with SaCas9 expression. B, Representative confocal images of ventricle myocytes from R176Q/+ mice co-injected with Bbsl-SaCas9 (control) + “reporter” and gRNA-SaCas9 (editing) + “reporter”, merged with bright field (BF) and red fluorescence (RFP) signal overlay. Scale bar, 100 μm. C, Bar graph showing percentages of mCherry-positive cardiomyocytes. D-F, Summary data for Ca2+ spark frequency, SR Ca2+ load, and SR Ca2+ transient amplitude. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The AAV-CRISPR vector, 1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA was generated by the Lagor lab (Addgene plasmid 109320) based on the SaCas9 transgene from px602, Addgene plasmid 61593 (gift of Dr. Feng Zhang).

Techniques: Injection, Virus, Clone Assay, Sequencing, Control, Expressing, Fluorescence

Journal: Nature communications

Article Title: CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I.

doi: 10.1038/s41467-018-07827-1

Figure Lengend Snippet: Fig. 1 Efficient GO inhibition using CRISPR/Cas9 in PH1 animals. a Schematic representation of the CRISPR/Cas9-mediated SRT strategy targeting the Hao1 locus. b Editing efficiency measured by TIDE in 12–14-week-old PH1 animals 4 weeks after treatment with saline (n = 5), Cas9 (n = 5), Hao1-g1 (n = 5) and Hao1-g2 (n = 5). c Quantification of Hao1 mRNA expression levels by RT-qPCR in animals treated as in (b). Data are presented as mean ± SEM and Kruskal–Wallis statistical test was used to evaluate differences between groups. d Western blot analysis of GO protein levels in representative PH1 animals treated with saline, Cas9, Hao1-g1, and Hao1-g2. GAPDH was used as loading control. e Representative IHC images of liver sections stained for GO, from PH1 animals treated with saline, Cas9, Hao1-g1, and Hao1-g2. Scale bar: 100 μm. *p < 0.05

Article Snippet: The pX602-AAV-TBG::NLS-SaCas9NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA plasmid that contains the Staphylococcus aureus Cas9 (SaCas9) expressed under the tTBG promoter, the sgRNA under U6 promoter and ITR sequences for AAV vector production was a gift from Feng Zhang (Addgene plasmid # 61593)44. sgRNAs targeting exonic regions of the Hao1 gene were designed and selected using Benchling software (www.benchling. com).

Techniques: Inhibition, CRISPR, Saline, Expressing, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: Nature communications

Article Title: CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I.

doi: 10.1038/s41467-018-07827-1

Figure Lengend Snippet: Fig. 3 Therapeutic efficacy of CRISPR/Cas9-mediated STR in PH1 animals. a Schematic experimental procedure, where 8–10-week-old PH1 animals were intravenously treated with saline (n = 7), Cas9 (n = 6), Hao1-g1 (n = 6) and Hao1-g2 (n = 5). A 7-days EG challenge was performed 4 months after vector administration, and 24 h urine samples were collected before and on days 3 and 7 of challenge. b Quantification of basal urine levels of oxalate and glycolate (µmol/24 h) 4 months after treatment. c, d Quantification of urine oxalate (c) and glycolate (d) levels (µmol/24 h) before and on days 3 and 7 of EG challenge. Data are presented as mean ± SEM and Kruskal–Wallis statistical test was used to compare the groups in each day. e Weight change of the animals during a one-week EG challenge. f Representative histological analysis of CaOx accumulation in the kidneys of PH1 animals from control or treatment groups after a 10-days EG challenge. Scale bar: 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: The pX602-AAV-TBG::NLS-SaCas9NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA plasmid that contains the Staphylococcus aureus Cas9 (SaCas9) expressed under the tTBG promoter, the sgRNA under U6 promoter and ITR sequences for AAV vector production was a gift from Feng Zhang (Addgene plasmid # 61593)44. sgRNAs targeting exonic regions of the Hao1 gene were designed and selected using Benchling software (www.benchling. com).

Techniques: CRISPR, Saline, Plasmid Preparation, Control