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Updated: Aug 1, 2025
Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System
Published on: December 10, 2021
DNA double-strand break-free CRISPR interference delays Huntington's disease progression in mice
Jung Hwa Seo1,2, Jeong Hong Shin2,3, Junwon Lee4
1Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea.
Abstract:
Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. CRISPR-Cas9 nuclease causes double-strand breaks (DSBs) in the targeted DNA that induces toxicity, whereas CRISPR interference (CRISPRi) using dead Cas9 (dCas9) suppresses the target gene expression without DSBs. Delivery of dCas9-sgRNA targeting CAG repeat region does not damage the targeted DNA in HEK293T cells containing CAG repeats. When this study investigates whether CRISPRi can suppress mutant HTT (mHTT), CRISPRi results in reduced expression of mHTT with relative preservation of the wild-type HTT in human HD fibroblasts. Although both dCas9 and Cas9 treatments reduce mHTT by sgRNA targeting the CAG repeat region, CRISPRi delays behavioral deterioration and protects striatal neurons against cell death in HD mice. Collectively, CRISPRi can delay disease progression by suppressing mHtt, suggesting DNA DSB-free CRISPRi is a potential therapy for HD that can compensate for the shortcoming of CRISPR-Cas9 nuclease.
Insights
CRISPR interference (CRISPRi) offers a novel therapeutic approach for Huntington's disease (HD) by suppressing mutant huntingtin (mHTT) gene expression without causing DNA damage. This DNA-double-strand-break-free method shows promise in delaying disease progression and protecting neurons.
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene.
- CRISPR-Cas9 nuclease technology, while effective for gene editing, can induce toxic DNA double-strand breaks (DSBs).
- CRISPR interference (CRISPRi) utilizes a deactivated Cas9 (dCas9) to suppress gene expression without causing DSBs.
Purpose of the Study:
- To investigate the efficacy of CRISPRi in suppressing mutant huntingtin (mHTT) expression in Huntington's disease models.
- To evaluate the safety and therapeutic potential of CRISPRi as a DNA double-strand-break-free approach for HD.
- To compare the effects of CRISPRi and CRISPR-Cas9 on mHTT levels and disease progression in HD.
Main Methods:
- Delivery of dCas9-sgRNA targeting the CAG repeat region in HEK293T cells and human HD fibroblasts.
- Assessment of mHTT and wild-type HTT expression levels.
- Evaluation of behavioral changes and striatal neuron survival in HD mouse models following dCas9 and Cas9 treatments.
Main Results:
- CRISPRi successfully reduced mHTT expression while preserving wild-type HTT in human HD fibroblasts.
- Both dCas9 and Cas9 targeting the CAG repeat region reduced mHTT.
- CRISPRi treatment in HD mice delayed behavioral deterioration and protected striatal neurons from cell death.
Conclusions:
- CRISPRi is a viable DNA double-strand-break-free therapeutic strategy for Huntington's disease.
- CRISPRi effectively suppresses mHTT, delays disease progression, and offers neuroprotection in HD models.
- CRISPRi presents a potential advantage over CRISPR-Cas9 by avoiding DNA damage for HD therapy.
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