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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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Research Article Genetics Neuroscience Article has an altmetric score of 17

CRISPR/Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA

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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 2

Guided Cas13d specifically decreased translation associated with GGGGCC repeat–containing transcripts.

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Guided Cas13d specifically decreased translation associated with GGGGCC ...
(A) Schematic of the inducible luciferase-based C9orf72 RAN translation reporter system in HeLa Flp-In cells. (B) The reporter cells stably expressing Cas13d and gRNA S24 and S30 showed lower signals of NanoLuc and firefly luciferase signal from the (GGGGCC)70-containing reporter transcripts. The significant reduction of the NanoLuc luciferase signal relative to the firefly luciferase signal demonstrates that the repeat-associated RAN translation is inhibited by Cas13d-mediated S24 or S30 treatment. (C) The control cells harboring the reporter without the G4C2 repeat showed no effect of the Cas13d gRNAs on the translation of the reporters. (D) Immunoblot analysis of C9orf72 protein showed that the C9orf72 protein level was unaffected in HeLa RAN translation reporter cell lines stably expressing Cas13d-NT30, Cas13d-S24, and Cs13d-S30. (E) Transient cotransfection of CRISPR/Cas13d constructs with either the GA-frame, GP-frame, or GR-frame or the No-G4C2-repeat control construct in HEK293 cells showed that Cas13d-S24 and Cas13d-S30 significantly reduced both NanoLuc and firefly luciferase signals from the GA-, GP-, and GR-frame but not the negative No-G4C2-repeat control reporter compared with the non-targeting control Cas13d-NT30. (F) Immunoblot analysis of C9orf72 protein showed that the C9orf72 protein level was unaffected in HEK293 cells cotransfected with CRISPR/Cas13d and either the GA-frame, GP-frame, or GR-frame or the No-G4C2-repeat control construct. Data are presented as means ± SD of 3 or 4 biological replicates as indicated by the number of dots in each graph, and were analyzed with ordinary 1-way ANOVA with Dunnett’s multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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