Sample size. Test and control vectors were evaluated in at least five mice per group at each time point to ensure reproducibility. Sample sizes are noted in figure legends.

Outliers. All data are presented.

Selection of endpoints. We chose 3 and 8 weeks after dosing in neonatal mice as relative short- and long-term time points, respectively, to evaluate OTC expression and gene targeting efficiency.

Replicates. Each vector group was evaluated in multiple litters of male spfash pups. Transduction efficiency in each mouse was analyzed on at least five images. Plasma NH3 was assayed in duplicate. Indel and HDR analysis on each sample was performed once.

Research objectives. This study aimed to develop a mutation-independent CRISPR-Cas9–mediated gene targeting approach in which the vector system could be applied to most patients with OTCD.

Research subjects. Newborn (p2) male spfash mouse pups were the subjects of this study. Untreated WT and spfash hemizygous mice served as controls.

Experimental design. Pups received a temporal vein injection of a mixture of two vectors at the intended doses for each with a volume of 50 μl. Mice were euthanized at various time points after vector treatment, and liver samples were harvested for analyses. Mice were genotyped at weaning or at the time of necropsy to confirm genotype. For testing the efficacy of OTC gene targeting, a high-protein diet (40% protein; Animal Specialties and Provisions, Quakertown, PA) was given to 7-week-old mice for 7 days. After this time, plasma was collected for measurement of plasma NH3 using an Ammonia Assay Kit (Sigma-Aldrich, St. Louis, MO).

Randomization. Note that the entire litter of newborn male pups was injected with either the targeting or control vectors, and no specific randomization method was used.

Blinding. The following assays were performed in a blinded fashion, in which the investigator was unaware of the nature of the vectors or vector dose: vector injection, OTC immunostaining, OTC enzyme activity staining and quantification, OTC enzyme activity assay, and vector GC analysis.

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