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 (dGAG) in the TOR1A gene. This study demonstrates a gene editing approach that selectively targets the dGAG 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 neuroprecursor 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 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 Next Generation CRISPR sequencing results analysed by CRISPResso2 software to detect allele edits or lack thereof