Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model

DYT1 dystonia is a neurological movement disorder characterized by a dominant 3-base pair deletion (ΔGAG) in the TOR1A gene. This study demonstrates a gene-editing approach that selectively targets the ΔGAG mutation in the TOR1A DYT1 allele while safeguarding the wild-type (WT) TOR1A allele. We optimized an adeno-associated virus (AAV) vector-compatible variant of the Staphylococcus aureus Cas9 nuclease ortholog (SaCas9-KKH) in DYT1 patient-derived human neuronal progenitor cells (hNPCs). On-target editing of the TOR1A DYT1 allele was confirmed at the genomic level from brain tissue in a xenograft mouse model. To avoid brain biopsy for demonstrating TOR1A DYT1 editing, we developed a non-invasive monitoring method using extracellular RNA (exRNA). TOR1A exRNA was retrieved from the extracellular vesicle (EV) secretions of hNPCs and plasma samples, indicating whether the donor was a TOR1A DYT1 carrier. This technique enabled us to assess AAV-mediated disruption of the TOR1A DYT1 allele in the brains of mice using blood samples.