A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• The C9ORF72 gene's GGGGCC repeat expansion is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
• Disease pathogenesis involves toxic gain-of-function from transcribed repeat-containing RNA.
• Existing therapeutic strategies face challenges in specificity and efficacy.

Purpose of the Study:

• To develop a high-fidelity RNA-targeting CRISPR system for C9ORF72-linked ALS and FTD.
• To evaluate the system's efficacy in reducing toxic RNA species and downstream pathological hallmarks.
• To assess the system's safety and specificity in a relevant preclinical model.

Main Methods:

• Engineered a modified Cas13 enzyme for high-fidelity RNA targeting.
• Delivered the CRISPR system to the brain of a transgenic rodent model of C9ORF72 ALS/FTD.
• Analyzed changes in G4C2 repeat RNA expression, RNA foci, dipeptide repeat protein production, and transcriptional deficits.

Main Results:

• The Cas13-based system effectively reduced G4C2 repeat RNA expression without impacting normal C9ORF72 levels.
• Targeting led to decreased RNA foci formation and reduced dipeptide repeat protein production.
• Transcriptional deficits were reversed, and the system demonstrated improved transcriptome-wide specificity compared to its native form.

Conclusions:

• A high-fidelity RNA-targeting CRISPR system offers a promising therapeutic strategy for C9ORF72-linked ALS and FTD.
• This technology effectively targets toxic RNA species and ameliorates key disease pathologies in a preclinical model.
• The findings support the future development of RNA-targeting CRISPR therapies for these devastating neurodegenerative disorders.