Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function.

Area of Science:

• Genetics
• Molecular Biology
• Neuroscience

Background:

• DYT1 dystonia is primarily caused by a specific mutation in the TOR1A gene.
• This mutation leads to a dominant-negative effect, impairing normal torsinA protein function.
• Reducing the amount of mutant torsinA is a proposed strategy to restore normal torsinA activity.

Purpose of the Study:

• To investigate the efficacy of CRISPR-Cas9 gene editing in disrupting the mutant TOR1A allele in DYT1 patient fibroblasts.
• To assess the impact of disrupting the mutant allele on torsinA protein levels and function.
• To evaluate the potential for phenotypic recovery in DYT1 patient cells following gene editing.

Main Methods:

• Utilized an engineered Streptococcus pyogenes Cas9 variant (SpCas9-VRQR) for targeted gene editing.
• Applied CRISPR-Cas9 to selectively disrupt the c.907_909delGAG mutation in the TOR1A gene.
• Employed non-homologous end joining (NHEJ) for gene editing, leading to premature stop codons and truncated torsinA.

Main Results:

• Achieved highly efficient and selective targeting of the DYT1 allele.
• Generated truncated torsinA proteins (delta 302-332 aa) predicted to be functionally inactive.
• Observed a significant reduction in mutant torsinA protein levels in edited fibroblasts.
• Demonstrated phenotypic recovery in a functional assay (HSV infection) towards control levels.

Conclusions:

• Selective disruption of the mutant TOR1A allele via CRISPR-Cas9 effectively inactivates mutant torsinA.
• This inactivation allows the remaining wild-type TOR1A allele to function normally, potentially correcting the DYT1 dystonia phenotype.
• CRISPR-Cas9-mediated gene editing presents a promising therapeutic avenue for DYT1 dystonia.