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Hum Mol Genet. 2016 Oct 15;25(20):4566-4576. doi: 10.1093/hmg/ddw286.

Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9.

Shin JW1,2, Kim KH1,2, Chao MJ1,2, Atwal RS1,2, Gillis T1, MacDonald ME1,2,3, Gusella JF1,3,4, Lee JM1,2,3.

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Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.
Department of Neurology, Harvard Medical School, Boston, MA, USA
Medical and Population Genetics Program, The Broad Institute of M.I.T. and Harvard, Cambridge, MA, USA
Department of Genetics, Harvard Medical School, Boston, MA, USA


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 the 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.

[Indexed for MEDLINE]
Free PMC Article

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