Primary hyperoxaluria type 1 (PH1) is a rare metabolic disease characterized by the overproduction of oxalate in the liver and its toxic accumulation in the kidneys. Currently, the best therapeutic option for patients with severe PH1 consists of reducing the expression of the glycolate oxidase (GO) enzyme by RNA interference. For a permanent disruption of GO, we investigated the use of paired Staphylococcus aureus Cas9 nickases (D10ASaCas9) targeting the Hao1 gene in a mouse model of the disease. We optimized an AAV-all-in-one construction to efficiently deliver D10ASaCas9 and the two gRNAs while minimizing the dose of vector administered. Double nicks resulted in heterogeneous modifications on-target, primarily repaired by the microhomology-mediated end joining (MMEJ) repair pathway.  As a result, GO expression was significantly reduced and oxalate overproduction was decreased. Moreover, no alteration in GO protein expression was measured when using D10ASaCas9 in combination with a single gRNA, supporting the safety of the strategy. Interestingly, the use of paired D10ASaCas9 resulted in a significant reduction of AAV integration events compared to those measured using the SaCas9 nuclease either combined with one or two gRNAs. The safety of the treatment was further evaluated by analyzing off-target modifications and chromosomal translocations using CAST-seq, and no such alterations were detected. Overall, these findings suggest that paired nickases targeting Hao1 represent a promising and safe long-term treatment for PH1 patients.