Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases. In this study, we established a novel all-in-one high-capacity adenovirus (HCAd) that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis type VII (MPS VII) caused by mutations in the β-glucuronidase (GUSB) gene. This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene (mActb) locus and the co-expression of GUSB with the self-cleaving peptide T2A (T2A) controlled by a strong endogenous mActb promoter. The in vivo results indicated that the serum GUSB level of MPS VII mice treated with a single intraperitoneal injection of the HCAd vector achieved 14% of that of wild-type mice, resulting in significant amelioration of lysosomal storage in the liver and spleen. Furthermore, the HCAd was injected intraventricularly in the brain of newborn MPS VII mice, leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage. In summary, by using an all-in-one vector, the study provides a universal, one-for-all therapeutic for MPSVII, a disease caused by different mutations of the GUSB gene.