PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease
Jun Wan Shin1,2, Eun Pyo Hong1,2, Seri S Park1
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
Insights
CRISPR-Cas9 gene editing offers a potential therapy for Huntington's disease (HD) by selectively targeting the mutant huntingtin (HTT) gene. This approach utilizes specific genetic markers to inactivate the disease-causing allele without affecting the healthy one.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Neurodegenerative Diseases
Background:
- Huntington's disease (HD) is a dominant neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene.
- Therapeutic strategies for HD must selectively inactivate the mutant HTT allele while preserving the function of the normal HTT allele, as complete HTT loss causes neurological issues.
- Previous research proposed using CRISPR-Cas9 technology with mutant-specific PAM sites, generated by single nucleotide polymorphisms (SNPs), for selective HTT inactivation.
Purpose of the Study:
- To identify suitable targets for clinical development of CRISPR-Cas9-based Huntington's disease therapies.
- To analyze the largest Huntington's disease genotype dataset to find target PAM-altering SNPs (PAS).
- To evaluate the allele specificity of identified PAS for selective mutant HTT inactivation.
Main Methods:
- Analysis of a comprehensive Huntington's disease genotype dataset to identify PAM-altering SNPs (PAS).
- Design and evaluation of guide RNAs (gRNAs) targeting PAM sites generated by specific SNPs (rs2857935, rs16843804, rs16843836).
- Experimental validation of allele specificity in patient-derived cells, including genomic deletion analysis, mRNA transcription assessment, RNA-sequencing, and off-target analysis.
Main Results:
- gRNAs targeting PAM sites generated by rs2857935, rs16843804, and rs16843836 demonstrated high allele specificity in patient cells.
- Simultaneous use of dual gRNAs (e.g., rs2857935-rs16843804) resulted in selective genomic deletions of mutant HTT and prevented mutant HTT mRNA transcription.
- The strategy did not impact normal HTT expression, showed no re-integration of excised fragments, and confirmed high specificity with no recurrent off-target mutations via RNA-seq.
Conclusions:
- PAM-altering SNPs provide effective targets for allele-specific CRISPR-Cas9 strategies in Huntington's disease.
- The identified PAS (rs2857935, rs16843804, rs16843836) enable selective inactivation of mutant HTT without affecting the normal allele.
- Approximately 60% of HD patients are eligible for this PAS-based CRISPR-Cas9 approach, indicating broad clinical applicability.
Abstract:
Huntington's disease (HD) is caused by an expanded CAG repeat in huntingtin (HTT). Since HD is dominant and loss of HTT leads to neurological abnormalities, safe therapeutic strategies require selective inactivation of mutant HTT. Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant HTT. Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to identify target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant HTT and prevented the transcription of mutant HTT mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNA-seq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these three PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population.
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