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 Overall design: Next Generation CRISPR sequencing results analysed by CRISPResso2 software to detect allele edits or lack thereof