Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9
| Autor: | Kyung Hee Kim, Ranjit Singh Atwal, Jong-Min Lee, Tammy Gillis, James F. Gusella, Jun-Wan Shin, Michael J. Chao, Marcy E. MacDonald |
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| Rok vydání: | 2016 |
| Předmět: |
Male
0301 basic medicine Huntingtin Mutant Single-nucleotide polymorphism Biology Polymorphism Single Nucleotide Cell Line 03 medical and health sciences Genetics Humans CRISPR Precision Medicine Allele Molecular Biology Alleles Genetics (clinical) Huntingtin Protein Cas9 Articles Genetic Therapy General Medicine Fibroblasts Middle Aged Molecular biology Protospacer adjacent motif Huntington Disease 030104 developmental biology Haplotypes Mutation CRISPR-Cas Systems Trinucleotide Repeat Expansion Trinucleotide repeat expansion |
| Zdroj: | Human Molecular Genetics. :ddw286 |
| ISSN: | 1460-2083 0964-6906 |
| Popis: | A comprehensive genetics-based precision medicine strategy to selectively and permanently inactivate only mutant, not normal allele, could benefit many dominantly inherited disorders. Here, we demonstrate the power of our novel strategy of inactivating the mutant allele using haplotype-specific CRISPR/Cas9 target sites in Huntington's disease (HD), a late-onset neurodegenerative disorder due to a toxic dominant gain-of-function CAG expansion mutation. Focusing on improving allele specificity, we combined extensive knowledge of huntingtin (HTT) gene haplotype structure with a novel personalized allele-selective CRISPR/Cas9 strategy based on Protospacer Adjacent Motif (PAM)-altering SNPs to target patient-specific CRISPR/Cas9 sites, aiming at mutant HTT allele-specific inactivation for a given diplotype. As proof-of-principle, simultaneously using two CRISPR/Cas9 guide RNAs (gRNAs) that depend on PAM sites generated by SNP alleles on the mutant chromosome, we selectively excised ~44 kb DNA spanning promoter region, transcription start site, and the CAG expansion mutation of the mutant HTT gene, resulting in complete inactivation of the mutant allele without impacting the normal allele. This excision on the disease chromosome completely prevented the generation of mutant HTT mRNA and protein, unequivocally indicating permanent mutant allele-specific inactivation of the HD mutant allele. The perfect allele selectivity with broad applicability of our strategy in disorders with diverse disease haplotypes should also support precision medicine through inactivation of many other gain-of-function mutations. |
| Databáze: | OpenAIRE |
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