Direct Targeting the Expanded CAG Repeat in Huntington's Diseases Using CRISPR/Cas9

Huntington's diseases (HD) is a dominantly inherited neurodegenerative disease, caused by an expanded CAG trinucleotide repeat in the first exon of huntingtin gene (HTT). The length of the repeat length, which is highly polymorphic in the general population, determine one's lifetime risk of developing characteristic HD symptoms such as involuntary movement, progressive decline of cognitive functions, and various psychiatric changes. People with 36-39 HTT CAG repeats show reduced penetrance; HTT CAG repeats longer than 39 cause fully penetrant movement disorder and neurological changes. In addition, the size of expanded repeat shows a robust inverse correlation with age at onset, indicating the size of the CAG repeat is the most important determinant of the disease. Therefore, inactivation of the mutation may represent the most efficient approach to delay the disease onset and to ameliorate clinical symptoms of this dominant disease.

Aiming at developing novel HD therapeutic strategies, we tested CRISPR/Cas9 genome editing approaches to directly target the HTT CAG repeat to inactivate the disease-causing mutation in HD. We screened various HTT CAG repeat-targeting CRISPR gRNAs in order to identify CRISPR strategies with therapeutic potential. Specifically, Mi-seq sequencing analysis was performed to determine targeting efficiencies and allele specificities of CRISPR gRNAs in HEK293 cells and induced pluripotent stem cells (iPSC) derived from HD subjects. Our data supported that directly targeting the HTT CAG repeat is feasible in HD, informing therapeutic strategies to permanently inactivate the root cause of the disease using CRISPR system to intervene in HD pathogenesis.

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