Metabolic rewiring yields similar outcomesin both female and male fah−/− miceUp to this point in the study, only male mice were used eventhough sexual dimorphism in response to similar rAAV-basedgenetic treatments had previously been reported (Davidoff et al.2003; Rinn and Snyder 2005; Paulk et al. 2012a). Reassuringly,others have shown that inactivation of Hpd using CRISPR-Cas9corrects the lethal HT-I phenotype in both female and maleFah−/− animals when delivered as plasmids via hydrodynamictail vein injections or rAAV (Pankowicz et al. 2016; Ibraheim et al.2018). Still, the impact of liver-specific inactivation of Hgd andGstz1 via in vivo genome editing in female Fah−/− animals hasyet to be reported. To better account for sex as a biological variableand improve the quality of our study, we targeted Hgd and Gstz1 inneonatal female and male Fah−/− pups side-by-side and monitoredtreatment outcomes. Upon NTBC withdrawal, mice of both sexesexperienced sudden weight loss and hypoglycemia and met theweight loss criterion within 4 days (Fig. 3, a-c). We also observedenlarged and pale kidneys in female mice treated with theHgd-targeting vector (Fig. 3d) like those observed in male Fah−/−mice treated with the same vector (Fig. 5b). In addition, gene dis-ruption efficacy was equivalent in both groups indicating that invivo CRISPR-Cas9 based editing using rAAV8 is similarly effica-cious in our model system (Fig. 3e). Thus, liver-specific inactiva-tion of either Hgd or Gstz1 by rAAV8-SaCas9 yields similarphenotypic outcomes in both female and male Fah−/− mice.

Standard care includes diet therapyto limit phenylalanine and tyrosine intake and lifelong treatmentwith 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione(NTBC; also known as nitisinone), a potent inhibitor of the up-stream enzyme HPD (Alvarez et al. 2017), to prevent toxic metab-olite accumulation in the liver and kidneys [Grompe et al. 1995;Lindstedt et al. 1992; Larochelle et al. 2012 (Fig. 1a)]. Importantly,non-compliance with NTBC and diet treatment is a serious chal-lenge for the clinical management of HT-I and results in higherrisks of patients developing hepatocellular carcinoma, as well aspainful corneal lesions due to high circulating tyrosine levelsand neurological crises due to high SA levels (