Collagen type VI is a microfibrillar collagen composed of three alpha chains [a1(VI), a2(VI), and a3(VI)], which are encoded by COL6A1, COL6A2, and COL6A3, respectively. Mutations in these chains are known to cause collagen VI-related dystrophies, which have a range of phenotypic severity from the milder Bethlem myopathy to the severe Ullrich congenital muscular dystrophy (UCMD). Recently, our group and others identified a de novo heterozygous deep-intronic variant (c.930+189C>T) in intron 11 of the COL6A1 gene associated with UCMD. This variant creates a donor splice site and, consequently, activates a pathogenic dominantly acting pseudoexon insertion. We generated a Col6a1 humanized knock-in mouse modeling the pseudoexon insertion and enabling the testing of exon-skipping approaches. Here, we aim at using a CRISPR/Cas9 and a dual guide RNA (gRNA) approach to excise the genomic sequence encoding the pseudoexon by targeting the flanking introns with gRNAs, and to ultimately deliver this technology systemically in vivo to mice with adeno-associated virus (AAV). We designed a pair of gRNAs flanking the pseudoexon to excise a 103-bp of genomic sequence. Patient-derived fibroblasts were nucleofected with plasmids carrying both SpCas9 and the two gRNAs, or carrying only SpCas9 as a negative control. Next, we prepared a dual AAV2 system expressing SpCas9 promoter and the two gRNAs, respectively, that we delivered to patient-derived fibroblasts. Similarly, 6-week-old mice carrying the COL6A1 pseudoexon insertion were treated via tail vein injection at 1.5E14 vg/kg with a dual AAV system. Tissues were collected 4 weeks later. Genomic DNA and RNA were isolated from in vitro and in vivo experiments and analyzed using PCR to assess editing efficiency. We successfully detected the 103-bp genomic excision in patient fibroblasts and mice following viral delivery of SpCas9 and dual gRNAs. In patient fibroblasts, higher editing efficiency was observed when dual gRNAs were delivered at a 3:2 ratio compared to Cas9. In mice, low levels (1% – 8%) of editing were observed in the gastrocnemius and quadriceps, while liver and heart tissue demonstrated higher editing efficiencies (4.5% – 28.0%). Viral delivery of CRISPR/Cas9 offers a potential therapeutic strategy for Collagen VI-related dystrophies. Editing efficiency remains a challenge and ongoing work is thus dedicated to address this.