CRISPR/Cas9 does not facilitate stable expression of long C9orf72 dipeptides in mice

Area of Science:

• Neuroscience
• Genetics

Background:

• The C9orf72 repeat expansion is a primary genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND).
• This expansion produces toxic dipeptide repeat proteins (DPRs), but the mechanisms of toxicity are not fully understood.
• Developing mouse models that accurately reflect human disease repeat lengths is crucial for mechanistic studies.

Purpose of the Study:

• To generate transgenic mice expressing long C9orf72 repeat expansions (>1000 repeats) using alternative codon sequences.
• To assess the stability of these long repeat sequences across generations in a mouse model.
• To explore alternative methods for delivering long repeat sequences for disease modeling.

Main Methods:

• Transgenic mice were created using alternative codon sequences to mitigate the repetitive nature of the C9orf72 repeat expansion insert.
• The stability of the integrated repeat sequences was evaluated across successive generations.
• The potential of viral vector administration as an alternative delivery method was considered.

Main Results:

• While the alternative codon inserts integrated into the mouse genome, they did not prevent instability between generations.
• The long DPR sequences exhibited instability, suggesting challenges with stable germline transmission.
• These findings indicate that stable integration of very long repeat sequences via traditional transgenesis may be unfeasible.

Conclusions:

• Stable integration of long C9orf72 repeat expansions in mice using current transgenesis methods is difficult.
• Alternative codon sequences do not confer stability to these long repeats.
• Viral vector-mediated delivery after birth may be a more promising approach for modeling long repeat expansions in FTD and MND.