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Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders
Fábio Duarte1,2, Gabriel Vachey1,2, Nicholas S Caron3
1Laboratory of Cellular and Molecular Neurotherapies, Department of Clinical Neurosciences (DNC).
Insights
CRISPR/Cas9 gene editing can inactivate mutant huntingtin (mHTT) by deleting exon 1. Dual sgRNA strategies efficiently deleted mHTT exon 1 in cells (67%) but showed lower deletion rates in mice (10%).
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic CAG expansion in the huntingtin (HTT) gene.
- Inactivating the mutant HTT (mHTT) allele is a promising therapeutic strategy due to HD's monogenic nature.
- CRISPR/Cas9 systems targeting single nucleotide polymorphisms near CAG expansions offer allele-selective mHTT inactivation.
Purpose of the Study:
- To quantify the frequency of HTT exon 1 deletion induced by dual sgRNA strategies.
- To assess the efficiency of this approach in vitro (HEK293T cells) and in vivo (humanized HU97/18 mice).
Main Methods:
- Development of quantitative digital PCR assays for precise HTT exon 1 deletion assessment.
- Application of dual sgRNA CRISPR/Cas9 strategies in HEK293T cells and HU97/18 mice.
- Analysis of editing outcomes in relation to CAG expansion and gene copy number.
Main Results:
- Dual sgRNA strategies efficiently induced HTT exon 1 deletion in HEK293T cells, with 67% of editing events resulting in deletion.
- In HU97/18 mice, while HTT cleavage occurred, exon 1 deletion rates were significantly lower (10%).
- In vivo editing patterns were not influenced by CAG expansion but potentially by multiple wildtype/mutant HTT gene copies and slow AAV delivery kinetics.
Conclusions:
- Dual sgRNA-mediated HTT exon 1 deletion is an effective strategy for mHTT inactivation in vitro.
- Significant differences in deletion efficiency between in vitro and in vivo models highlight challenges for in vivo therapeutic applications.
- Further research is needed to optimize CRISPR/Cas9 delivery and editing efficiency in vivo for Huntington's disease treatment.
Abstract:
Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.
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