CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington's Disease

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a mutation in the Huntingtin gene (HTT).
• The mutation leads to the production of toxic mutant huntingtin proteins (mHTT) with expanded poly-glutamine repeats.
• Current therapeutic strategies aim to reduce mHTT production.

Purpose of the Study:

• To develop and test CRISPR-Cas9 gene editing strategies to curb mHTT production.
• To evaluate the efficacy of targeting specific DNA regions within the HTT gene.

Main Methods:

• Construction of two CRISPR-Cas9 plasmids targeting the HTT gene.
• Application of CRISPR-Cas9 plasmids into mesenchymal stem cells (MSCs) from YAC128 HD mouse models.
• Analysis of DNA modifications and mHTT translation post-editing.

Main Results:

• Disruption of the untranslated region (uORF) via CRISPR-Cas9 significantly reduced mHTT translation.
• Disruption of the exon1-intron boundary also affected mHTT translation, though to a lesser extent.
• Characterization of nucleotide addition/deletion patterns at CRISPR-Cas9 targeted sites.

Conclusions:

• CRISPR-Cas9 targeting of the HTT gene, particularly the uORF, is a viable strategy to reduce mHTT production.
• This approach holds potential for developing novel therapies for Huntington's disease.
• Understanding DNA repair mechanisms post-editing is crucial for optimizing gene editing outcomes.