CRISPR/Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA
Honghe Liu, … , Lindsey R. Hayes, Jiou Wang
Honghe Liu, … , Lindsey R. Hayes, Jiou Wang
Published September 17, 2024
Citation Information: J Clin Invest. 2024;134(21):e179016. https://doi.org/10.1172/JCI179016.
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CRISPR/Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA

Abstract

A hexanucleotide GGGGCC repeat expansion in the non-coding region of the C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR/Cas13d, a powerful RNA-targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR/Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR/Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR/Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.

Authors

Honghe Liu, Xiao-Feng Zhao, Yu-Ning Lu, Lindsey R. Hayes, Jiou Wang

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Figure 5

The CRISPR/Cas13d system suppressed RAN translation in C9orf72 repeat transgenic mice.

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The CRISPR/Cas13d system suppressed RAN translation in C9orf72 repeat tr...
(A) Schematic of poly-GP and poly-GA detection in mice treated with AAV9 expressing Cas13d and gRNA. (B and C) Quantification showed decreased levels of poly-GP (B) or poly-GA (C) in C9-500 BAC mice but not in the WT mice, when the mice were treated with AAV9 expressing Cas13d-S24 or Cas13d-S30, compared with those treated with control AAV9 expressing the non-targeting Cas13d-NT30. The number of dots in each group indicates the number of mice in the corresponding group. Data are presented as means ± SD and were analyzed with unpaired 1-tailed Student’s t test. *P < 0.05, ****P < 0.0001.