Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9.
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.
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Neurology, Harvard Medical School, Boston, MA, USA
Jun Wan
JW
Shin
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Neurology, Harvard Medical School, Boston, MA, USA
Kyung-Hee
KH
Kim
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Neurology, Harvard Medical School, Boston, MA, USA
Michael J
MJ
Chao
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Neurology, Harvard Medical School, Boston, MA, USA
Ranjit S
RS
Atwal
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.
Tammy
T
Gillis
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
Marcy E
ME
MacDonald
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, 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
James F
JF
Gusella
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
Jong-Min
JM
Lee
D064113:CRISPR-Cas Systems; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D006239:Haplotypes; D006801:Humans; D000071058:Huntingtin Protein / Q000235:genetics*; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D057285:Precision Medicine / Q000379:methods; D019680:Trinucleotide Repeat Expansion
C086055:HTT protein, human; D000071058:Huntingtin Protein