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

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related ‘modifier’ genes (i.e., genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.


INTRODUCTION
Inborn errors of metabolism (IEMs) are inherited genetic disorders caused by disruptions in a specific enzymatic reaction within a metabolic pathway that induce pathology through toxic metabolite accumulation or deficiencies in downstream metabolites 1 .First described by Archibald Garrod over 100 years ago, IEMs are frequently considered to be monogenic diseases 2 .However, this classification may over-simplify the biological reality, as patients often present a spectrum of phenotypes [3][4][5] .Importantly, modifier genes can profoundly influence the phenotype associated with mutation(s) (pathogenic variants) at a primary "disease-causing" gene locus [3][4][5][6][7][8] .Genome editing therapies targeting modifier genes are already showing great promise in clinical trials, particularly for hemoglobinopathies [9][10][11] .
Liver-specific base editing of PCSK9 is also currently being investigated in a clinical trial to treat familial hypercholesterolemia 12,13 .Thus, identifying and characterizing modifier genes can provide valuable mechanistic information and spark the development of novel therapeutics.
The phenylalanine and tyrosine degradation pathway is notable historically as it is linked to the description of the first inborn error of metabolism, alkaptonuria 14 .Loss-of-function of each enzyme in this pathway leads in a different IEM (Figure 1A).In the first step, phenylalanine is converted to tyrosine by phenylalanine hydroxylase (PAH).Loss of PAH activity leads to phenylketonuria; a disease characterized by intellectual disability and seizures (OMIM 261600).Tyrosine is then converted to 4-hydroxyphenylpyruvate (4-HPP) by tyrosine aminotransferase (TAT).TAT inactivation results in tyrosinemia type II, which causes painful corneal lesions, skin disease, and intellectual disability (OMIM 276600).In the next step, 4-HPP is converted to homogentisic acid (HGA) by 4hydroxyphenylpyruvate dioxygenase (HPD).Its loss-of-function results in tyrosinemia type III, a disease characterized by intellectual disability, seizures, and intermittent ataxia (OMIM 276710).In the fourth reaction, HGA is converted to maleylacetoacetate (MAA) by homogentisate 1,2-dioxygenase (HGD).Patients with inactive HGD display high HGA levels and develop alkaptonuria, the prototypical IEM described by Garrod 2 , which results in arthritis, heart valve and kidney issues, and pigmentation changes to the cartilage and urine (OMIM 203500).In the penultimate step in tyrosine degradation, MAA is converted to fumarylacetoacetate (FAA) by glutathione S-transferase zeta 1 (GSTZ1), also known as maleylacetoacetate isomerase (MAAI).Individuals with GSTZ1 deficiency display mild hypersuccinylacetonemia which appears to be clinically insignificant 15,16 (OMIM 617596).
Finally, fumarylacetoacetate hydrolase (FAH) converts FAA into fumarate and acetoacetate, which are used for energy production by the tricarboxylic acid cycle and reconverted to acetyl-CoA respectively.Among IEMs affecting this pathway, one of the most severe is undoubtedly hereditary tyrosinemia type 1 (HT-I), a disorder caused by the absence of a functional FAH allele (OMIM 276700) 17 .Loss of FAH activity can lead to hepatic failure with renal and neurological comorbidities, along with a high risk of developing hepatocellular carcinoma 18,19 .HT-I results from the accumulation of tyrosine and its toxic metabolites, such as FAA, MAA, and succinylacetone (SA), a by-product of both FAA and MAA degradation and the diagnostic marker for HT-I 20 .However, the molecular mechanisms by which these compounds damage the liver and kidneys are poorly characterized 18,20 .HT-I is progressive and life-threatening if left untreated.Standard care includes diet therapy to limit phenylalanine and tyrosine intake and lifelong treatment with 2-(2-nitro-4trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; also known as nitisinone), a potent inhibitor of the upstream enzyme HPD 21 , to prevent toxic metabolite accumulation in the liver and kidneys [22][23][24] (Figure 1A).Importantly, non-compliance with NTBC and diet treatment is a serious challenge for the clinical management of HT-I and results in higher risks of patients developing hepatocellular carcinoma, as well as painful corneal lesions and neurological crises due to high circulating tyrosine levels 25 .While this catabolic pathway is well characterized at the molecular genetic level, the broad spectrum of phenotypes and disease severity observed in patients with HT-I-even within families-is not completely understood 26 .
Several knockout mouse models have been created to evaluate the interplay between the different genes in the phenylalanine and tyrosine degradation pathway and their impacts on various disease phenotypes [27][28][29][30][31] .The Fah Δexon5 (Fah -/-) mouse is a well-established murine model for HT-I pathophysiology 23,27 .In these mice, neonatal lethality can be prevented by early treatment with NTBC, which completely corrects the metabolic liver disease and results in a phenotype analogous to human tyrosinemia type I 23 .Upon NTBC withdrawal, however, disease progression resumes leading to death in a short period of time 32 .This experimental system offers an opportunity to test treatment options in vivo.
Accordingly, both short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9directed inactivation of Hpd in the liver of Fah -/-animals enabled the survival after NTBC withdrawal, suggesting that metabolically blocking the pathway in the liver is sufficient for a systemic therapeutic effect [39][40][41][42] .Still, the impact of liver-specific inactivation of Hgd and Gstz1 via in vivo genome editing in the HT-I mouse model has yet to be described.To test this, we used rAAV8-mediated delivery of Streptococcus aureus Cas9 (SaCas9) 43 to inactivate Hpd, Hgd, and Gstz1 in neonatal Fah Δexon5 (Fah -/-) mouse (Figure 1A).We did not target Tat, as its loss produces a more severe phenotype in mice than the loss of Hpd, Hgd, and Gstz1, according to phenotyping data from the the International Mouse Phenotyping Consortium 27,30,31,38,44 .We found that, while targeting Hpd and Gstz1 yielded outcomes similar to those observed in whole-body double mutant mice, targeting Hgd led to systemic and lethal effects in contrast to the genetic suppression observed in the conventional Fah -/-Hgd -/-model.This work highlights the value of somatic genome editing in animals for modelling human disorders 45 .

SaCas9.
Since most of the enzymes in the tyrosine catabolic pathway are expressed mainly in the liver and kidneys, we hypothesized that liver-specific gene disruption would produce a systemic impact on metabolic functions.This can be achieved in neonatal mice using CRISPR-Cas9 delivered via rAAV8 injected systemically to target hepatocytes 46 .In this system, the expression of a single guide RNA (sgRNA) targeting a specific gene is driven by the ubiquitous U6 promoter, and SaCas9 is expressed from a liver-specific thyroxinebinding globulin (TBG) promoter 43 (Figure 1B).Following target cleavage in mouse hepatocytes, DNA repair mainly occurs through the error-prone non-homologous end joining pathway (NHEJ), creating insertions and deletions (indels) that disrupts the target gene 47 .
Using CRISPOR 48 , we designed several SaCas9 sgRNAs against Hpd, Hgd, and Gstz1 (the gene encoding MAAI, Figure 1A) (Table S1).We selected sgRNAs that targeted essential protein domains and had few predicted off-target effects on the mouse genome to increase the likelihood of generating inactivating mutations [49][50][51] .We identified highly active sgRNAs for all three targets by transient transfection in mouse neuroblastoma cells (Figure S1).Nucleases with perfectly conserved target sites and protospacer adjacent motif (PAM) sequences between the mouse and human genomes were chosen for Hpd and Hgd but the Gstz1 target site differs by 2bp (Figure 1B).All-in-one rAAV vectors expressing both an sgRNA targeting Hpd, Hgd, or Gstz1 and hepatocyte-specific SaCas9 (via the TBG promoter) were constructed and produced as hepatotropic rAAV serotype 8 (rAAV8) vectors (Figure 1B) 43,[52][53][54] .Next, neonatal (2-day-old) male C57BL/6N mice were injected into the retro-orbital sinuses with 5×10 10 vector genomes (VGs) of rAAV8 targeting Hpd, Hgd, or Gstz1, then sacrificed 40 (Hpd and Hgd) or 77 (Gstz1) days later.Genomic wholeliver (Hpd, Hgd, and Gstz1) and kidney (Hgd and Gstz1) DNA was extracted, and Surveyor assays were used to determine indel frequencies 55 .Gene disruption was robust from mouse to mouse and generally efficient, ranging from 11-47% depending on the target in the liver (Figure 1C).Since genomic DNA was extracted from whole livers and gene editing was limited to the hepatocytes (which comprise ~70% of the liver's mass 56 ), the disruption efficiency is likely an underestimate.No editing was detected in the kidneys as expected when using rAAV8 and a liver-specific promoter to drive SaCas9 expression (Figure 1C).
We then repeated this experiment in 2-day-old male Fah -/-mice maintained on NTBC until sacrifice at 28 days old and observed similar levels of gene disruption as determined by the tracking of indels by decomposition (TIDE) assay 57 (Figure 1D).The indel profiles obtained with the TIDE assay indicated the presence of out-of-frame mutations likely to result in gene inactivation (Figure S1).These editing rates are similar to previous in vivo editing studies indicating that our system is fully functional in this context 43,58 .

In vivo inactivation of various enzymes in the tyrosine catabolic pathway differentially impacts metabolic outcomes and survival in HT-I mice
The impacts of liver-specific in vivo genome editing of Hpd, Hgd, and Gstz1 (the gene encoding MAAI, Figure 1A) in HT-I mice were then determined upon NTBC withdrawal.
Neonatal male Fah -/-mice treated with NTBC were injected at 2 days old with rAAV8-SaCas9 targeting Hpd, Hgd, or Gstz1 (or saline as a control), and NTBC was withdrawn between 4-7 weeks old (Figure 2A).As expected, all mice treated with the nuclease targeting Hpd displayed normal lifespans, weights, and glycemia (blood glucose levels) post-NTBC removal, while saline-treated animals were sacrificed ~3 weeks after NTBC removal, when meeting the weight loss criterion (Figure 2B-D).Glycemia and weight gains were normalized for over 14 weeks after NTBC withdrawal in Hpd-treated mice and they were kept alive without NTBC for 1 year (Figure S2, Table S2).In sharp contrast, Fah -/-mice treated with rAAV8-SaCas9 targeting either Hgd or Gstz1 experienced sudden weight loss and hypoglycemia and died approximately 3 and 5 days post-NTBC removal, respectively (Figure 2B-D).We measured urine succinylacetone (SA) and homogentisic acid (HGA) levels respectively 24 hours before NTBC removal and 34 days after NTBC removal (Hpd) or the predicted time of death (Hgd and Gstz1).Before NTBC removal, the urine SA levels of the different treatment groups were broadly similar (Figure 2E, Table S3).The SA levels of mice treated with the Hpd-targeting vector decreased post-NTBC removal to only slightly above the detection limit indicating that the phenotype was normalized (Figure 2E, Table S3).Notably, mice treated with the Gstz1-targeting vector showed marked increases in urine SA after drug withdrawal that were equal to or higher than saline treated controls (Figure 2E, Table S3).Urine HGA levels were undetectable in all animals except in mice treated with the vector targeting Hgd, which experienced a considerable increase following NTBC withdrawal (Figure 2F, Table S4).These biochemical changes correspond to the step blocked in the pathway (Figure 1A).TIDE assays on the livers of sacrificed mice treated with the vectors targeting Hgd or Gstz1 showed editing levels comparable to those seen in our previous tests with healthy animals (Figures 2G and 1C,D).Collectively, these data indicate that somatic genome editing can rewire the liver metabolism in HT-I mice to either suppress or enhance the disease.
As kidneys of HT-I mice are sensitive to cytotoxicity 23,27,30,59 , we performed SDS-PAGE analysis of urine samples and observed a massive increase in serum albumin (also known as albuminuria) in the animals treated with the vectors targeting Hgd and Gstz1 following NTBC removal, indicative of kidney disease 60 (Figure 3C).At harvest, the kidneys of mice treated with the Hgd-targeting vector were pale, enlarged, and displayed severe diffuse tubular damage with necrosis, which is consistent with HGA-induced toxic tubular cell injury leading to acute renal insufficiency (Figures 3A, top

Metabolic rewiring also occurs in "wild-type" mice
To ensure that the severely aggravated phenotypes observed in Hgd-and Gstz1-targeted animals were specific to the Fah -/-mouse model, we injected C57/Bl6N neonates with the three vectors or saline.We did not observe any changes in survival or glycemia between vector-treated animals and saline controls (Figure 4A,B).However, animals treated with the Gstz1-targeting vector had elevated urine SA levels, and animals injected with the Hgdtargeting vector had detectable HGA levels as expected from their respective metabolic blocks (Figure 4C,D and Tables S5,S6).Thus, this approach can partially recapitulate biochemical phenotypes associated with IEMs in wild-type animals, even when the metabolic block is limited to a single organ and not all hepatocytes are edited.

DISCUSSION
Coupling rAAV-mediated in vivo gene delivery with CRISPR-Cas9 systems is a robust and rapid method to study gene functions in the somatic tissues of mice 45,54,[61][62][63] .Here, we demonstrate that in vivo genome editing can modulate IEM-associated pathways to yield distinct phenotypes from those observed in whole-body knockout models.A limitation of this methodology is that gene disruption in somatic tissues is heterogeneous and incomplete, leaving a fraction of cells with altered functions that can affect the phenotype.
In Fah -/-mice, metabolic rewiring via in vivo editing recapitulated several hallmarks of HT-I.First, targeting Hpd rescued the lethal phenotype, as previously observed 37,[39][40][41] .This rescue was maintained for at least 1 year post-NTBC removal.An increase in the editing level occurred over time in these mice likely due to the potent growth advantage of corrected hepatocytes following NTBC removal 32,39,41 .We also observed that liverspecific targeting of Gstz1 in Fah -/-mice resulted in pronounced SA excretion and rapid death as in conventional double mutant Fah -/-Gstz1 -/-mice 30 (Figure 5).Interestingly, targeting Hgd resulted in the opposite phenotype compared to that observed in a classical knockout mouse model and when an FAH inhibitor was used to select Hgd -/-hepatocytes transplanted into wild-type recipient mice 38,64 .While whole-body Fah -/-Hgd -/-mice were protected from liver and renal damage, liver-specific inactivation of Hgd via in vivo editing in Fah -/-mice resulted in rapid death likely caused by kidney failure.This apparent discrepancy could be attributed to the fact that liver-specific Hgd disruption followed by NTBC withdrawal resulted in the rapid production of a massive amount of HGA, which is known to cause renal damage to Fah -/-mice 59,65 .This HGA can be processed in the kidneys by active HGD and MAAI leading to the local accumulation of FAA and SA since FAH is not present in this organ in the Fah -/-mice (Figure 5).Previous work had shown that phenotypic rescue of HT-I in mice can occur by Hgd inactivation, an in vivo suppressor mutation 38 .In these spontaneous revertants, liver-function tests were normal but kidneys were pale and enlarged and showed extensive tubular damage 38 .In this original work, these data were not shown but they are reminiscent of our observations.It appears that the main difference is that our system rapidly produced a higher fraction of hepatocytes inactivated for Hgd, that created a bolus of HGA following sudden NTBC removal, which prevented any adaptation and caused acute renal failure (Figure 5).In HT-I, there is evidence that negative feedback loops can inhibit or down-regulate upstream enzymes in the tyrosine degradation pathway.For example, in Fah -/-mice, TAT mRNA levels are markedly reduced without NTBC treatment 23 (Figure 1A).In humans, HPD's enzymatic activity is greatly reduced in patients with HT-I compared to healthy controls 17,66 .These compensatory mechanisms may partially protect liver and kidney cells from the toxic accumulation of FAA and SA and may have been bypassed by the sudden perturbations created by the editing process and NTBC withdrawal.
We propose a model of metabolic interplay between the edited liver and the non-edited kidneys in Fah -/-mice following NTBC withdrawal (Figure 5).Of note, this model does not exclude the possibility that circulating HGA can also re-enter non-targeted, HGDexpressing hepatocytes, and cause their rapid death 67,68 .Irrespective of the physiological mechanism, the differences between our observations and those of whole-body knockout models highlight the importance of using tissue-specific genome editing in animal models of human genetic disorders to investigate its systemic impacts.It has recently been shown that metabolic pathway rewiring following liver-directed CRISPR-Cas9 knockout can be used to rescue glutaric acidemia type 1, an inborn error of metabolism affecting the lysine degradation pathway 69 .In vivo genome editing is thus a promising approach that could be used to dissect metabolic pathways affected in other IEMs.
Other than the limitation imposed by the impossibility to edit all hepatocytes within the liver, even in mice, there may be major differences in phenotypes between humans and mice deficient in the same gene product.A prime example is that HT-I mice have a neonatal lethal phenotype and are not tyrosinemic (i.e. they do not have elevations of plasma tyrosine) 27 , unless treated with NTBC 23 , contrary to humans with tyrosinemia type I. Our studies have also been performed in neonates which may have differences in liver functions compared to adult mice.Finally, for historical reasons 52,53,70 , we only used male mice in this study even though sexual dimorphism in response to similar genetic treatments had been reported [71][72][73] .Reassuringly, we have observed that in vivo genome editing using Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) shows no such bias when targeting Hpd in this same mouse model 41 .

Genome editing vectors
The CMV-driven SaCas9 nuclease vector pX601 43 (Addgene plasmid #61591) was a gift from Feng Zhang (Massachusetts Institute of Technology).Target sequences for Hpd, Hgd, and Gstz1 were designed using the web-based CRISPR design tool CRISPOR 48 .The sgRNA sequences used are listed in Table S1.When required, the sgRNA sequence was modified to encode a G at position 1, to meet the transcription initiation requirement of the human U6 promoter.Following in vitro screening, selected SaCas9 sgRNAs were cloned into the TBG-driven SaCas9 nuclease rAAV vector pX602 43 (Addgene plasmid #61593; also a gift from Feng Zhang) for in vivo gene editing.The inverted terminal repeat integrity of the rAAV vector pX602 was assessed by BssHII digestion.

Surveyor and TIDE assays
Genomic DNA was extracted from 2.5×10 5 Neuro2A cells with 250 μL of QuickExtract DNA Extraction Solution (Lucigen, Middleton, WI, USA) or from 30 mg of mouse liver using a EZ-10 Spin Column Animal Genomic DNA Miniprep Kit (Bio Basic, Markham, ON, CA), per the manufacturers' recommendations.Loci were amplified by polymerase chain reaction (PCR) using the primers listed in Table S7.Surveyor assays were performed with the Surveyor Mutation Detection Kit (Integrated DNA Technologies, Coralville, IA, USA) as described 55 .Samples were resolved on 10% polyacrylamide gels in Tris-borate-EDTA buffer and bands were visualized with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Seongnam, South Korea).Gels were imaged using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) and bands were quantified using Image Lab Software (Bio-Rad).TIDE analysis was performed using a significance threshold value for decomposition of P < 0.001 57 .

Adeno-associated virus production
The rAAV8s were produced by the Canadian Neurophotonics Platform's Viral Vector Core (The Molecular Tools Platform) using the triple plasmid transfection method, as described 74 .Briefly, HEK293T17 (ATCC CRL-11268, Manassas, VA, USA) cells were transfected using polyethylenimine (Polysciences) with the helper plasmid pxx-680 (a gift from R. Jude Samulski, University of North Carolina), the rep/cap hybrid plasmid pAAV2/8 (Addgene #112864, a gift from James Wilson, University of Pennsylvania), and the rAAV vector pX602.After 24 hours, the medium was replaced with medium without FBS, and the cells were harvested 24 hours later.We purified rAAV particles from the cell extracts using freeze/thaw lysis followed by a discontinuous iodixanol gradient.Viruses were resuspended in phosphate-buffered saline containing 320 mM NaCl, 5% D-sorbitol, and 0.001% pluronic acid (F-68), aliquoted, and stored at -80°C.
The rAAVs were titrated by quantitative PCR using LightCycler 480 SYBR Green I Master mix (Roche, Basel, Switzerland) and inverted terminal repeat primers as described 75 .

Animal experiments
Fah -/-mice 27 in a C57BL/6 genetic background were a kind gift from Robert Tanguay (Laval University).C57BL/6N mice were purchased from Charles River (Strain code 027) (Wilmington, MA, USA).All mice were group-housed and fed a standard chow diet (Harlan #2018SX) with free access to food and water.For Fah -/-mice, the drinking water was supplemented with 7.5 mg/L NTBC.Mice were exposed to a 12:12-h dark-light cycle and kept at an ambient temperature of 23±1°C.Animals were cared for and handled according to the Canadian Guide for the Care and Use of Laboratory Animals.The Laval University Animal Care and Use Committee approved the procedures.
Neonatal (2-day-old) male mice were injected intravenously in the retro-orbital sinus 76 with saline or 5×10 10 VGs of an rAAV8, adjusted to an injection volume of 20 µL with saline.
Fah -/-mice were weaned at 21 days old and NTBC was removed at the indicated time points.Body weight and glycemia were monitored post-NTBC removal.Glycemia was measured in unfasted mice between 9 and 10 am.Animals were sacrificed by cardiac puncture under anesthesia at predetermined time points or when they had lost 20% of their body weight.The majority of the liver and one kidney were snap frozen, while a portion of the liver and the other kidney was fixed in 4% paraformaldehyde.

Urine collection and SDS-PAGE analysis
Urine was collected from groups of 2-5 mice overnight using metabolic cages (Tecniplast) before and at different time points after NTBC removal.Urine was centrifuged at 900 x g for 5 minutes, aliquoted, and frozen at -80°C for downstream applications.For SDS-PAGE analysis, 5µL of urine per mouse was loaded on a 4-15% Mini-PROTEAN TGX Stain-Free Gel (Bio-Rad, Hercules, CA, USA) before Coomassie staining with QC Colloidal Coomassie (Bio-Rad) and imaging using a ChemiDoc MP Imaging System (Bio-Rad).

Histology
Liver portions and kidneys were fixed in 4% paraformaldehyde for 24 hours, then dehydrated in 70% ethanol, embedded in paraffin, and sliced into 4 µm sections, which were mounted on slides.Liver sections were stained with hematoxylin and eosin and reviewed by a blinded pathologist and a blinded hepatologist at a magnification of 200X.Kidney sections were stained with Masson's trichrome and reviewed by a blinded nephrologist using an Olympus BX45 microscope at different magnifications.S3 and S4).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 Hgd-and Gstz1-targeting vectors meeting the sacrifice endpoint, and Surveyor assays were used to determine indel frequencies.Each symbol represents a different animal.A mouse injected with saline was used as the negative control for the Surveyor assay.S5 and S6).The detection limits for succinylacetone and homogentisic acid were 0.1 mmol/mol and approximately 3 mmol/mol creatinine, respectively.BDL: below detection limit.
panel and 3B).Those treated with the vector targeting Gstz1 had slight tubular damage mainly in the cortical area, similarly to saline-treated controls (Figure 3A, top panel).A group of Hpd-targeted mice sacrificed 35 days after NTBC withdrawal displayed normal tubules and glomeruli, suggesting preserved kidney function (Figure 3A, top panel).Liver histology revealed that mice injected with the Hgd-targeting vector displayed substantial hepatocyte death in zones 1 and 2 with signs of apoptosis, moderate bile ductular proliferation, and portal inflammation.Hepatic steatosis and fibrosis were not detected (Figure3A, bottom panel).In mice injected with the Gstz1-targeting vector, mild lobular inflammation and significant and diffuse ballooning degeneration indicative of apoptotic hepatocyte death was observed (Figure3A, bottom panel).In animals injected with the Hpd-targeting vector, liver sections displayed moderate portal inflammation, mild ductular proliferation, and mild ballooning degeneration in zones 2 and 3, suggesting that gene-edited hepatocytes had not completely repopulated the diseased liver at the time of necropsy (Figure3A, bottom panel).Finally, liver histology revealed mild portal and lobular inflammation, mild ductular proliferation, glycogenated nuclei, and mixed steatosis in zone 2 in saline-injected controls (Figure3A, bottom panel).Collectively, these data indicate that liver-based editing can cause systemic effects impacting kidney function.

Figure 1 |Figure 2 |
Figure 1 | In vivo genome editing of the tyrosine catabolic pathway by rAAV8-SaCas9 (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 C57/Bl6 mice 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 the following age: Hpd, 40 days; Hgd, 40 days; Gstz1, 77 days.Genomic whole-liver (Hpd, Hgd, and Gstz1) and kidney (Hgd and Gstz1) DNA was extracted, and Surveyor assays were used to determine indel frequencies.Each symbol represents a different animal.A mouse injected with saline was used as the negative control for the Surveyor assay.(D) Neonatal male Fah -/-mice were injected with 5×10 10 VGs of rAAV8-SaCas9 into the retro-orbital sinus, weaned at 21 days old, and sacrificed at 28 days old after continuous NTBC treatment.TIDE assays were used to determine the frequency of indels.A mouse injected with saline was used as the negative control for the TIDE assay.

5 Figure 3 |
Figure 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, or Gstz1 as in Figure 2. Fah -/-mice injected with saline and the Hpd, Hgd, and Gstz1 rAAV8s 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 (1µL per well) from treated Fah -/-animals on (+) and off (-) NTBC and untreated controls (as described in Figure2) were loaded on a 4-15% gradient mini-PROTEAN TGX Stain-Free gel before electrophoresis, Coomassie staining, and imaging.Urine from a C57/Bl6 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).

Figure 4 |
Figure 4 | C57/Bl6N mice treated with the rAAV8-SaCas9 vectors have partial metabolic phenotypes and normal survival (A) Survival analysis 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.The animals treated with the vectors targeting Hpd and Gstz1 are the same animals as in Figure 1C, whereas mice treated with the vector against Hgd are a separate group of animals injected solely for phenotypic studies.(B-D) Mice were assayed for phenotypic and metabolic modifications after weaning.(B) Glycemia of non-fasted mice.Solid lines indicate the mean and the shaded areas denote the SEM.Urine succinylacetone (C) and homogentisic acid (D) levels in mice treated as in (A) were determined 35 days after weaning.Samples were collected over 24 hours using metabolic cages containing 2-3 mice (TablesS5 and S6).The detection limits for succinylacetone and homogentisic acid were 0.1