Genetic foundation for complete mutant allele-specific CRISPR in neurodegenerative diseases

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SUMMARY / ABSTRACT Title: Genetic foundation for complete mutant allele-specific CRISPR in neurodegenerative diseases Many cases of dominant neurodegenerative disorders defy the development of effective treatments despite their long-known genetic causes and numerous implicated underlying pathways, reflecting difficulty in defining rational drug targets through investigations focusing on mechanisms. Alternatively, development and maturation of gene targeting/lowering approaches have highlighted the value of disease-causing mutation itself as the target of treatments. Although promising, mRNA-lowering approaches suffer from inherent limitations of requirement of repeated treatments, allele-specificity, and dosage controls. We recently developed a complete allele-specific DNA targeting strategy based on CRISPR gene editing technology using PAM-altering SNP to overcome key limitations of conventional mRNA targeting approaches. Our silencing strategy achieves perfect allele specificity by using SNP variations that create CRISPR PAM sites selectively on the mutant allele/haplotype. Importantly, our novel CRISPR gene silencing strategy targets the haplotype backbone that carries the disease-causing mutation, and therefore does not depend on the type, size, and location of the disease allele, providing broadly applicable therapeutic platforms. Here, we propose to determine therapeutic potential of PAM-Altering SNP (PAS)-based CRISPR strategies 1) to prevent the transcription or 2) to induce nonsense-mediated decay of the mutant allele in cells and animal models of Huntington's disease (HD). Briefly, we will: 1) determine whether mutant allele-specific Transcription Prevention by CRISPR (TP-CRISPR) and Nonsense-Mediated Decay by CRISPR (NMD-CRISPR) efficiently introduce knockout mutations in neuronal cells derived from patients and brains of animal models of HD, 2) test whether selective silencing of mutant allele of a developmentally important gene (i.e., HTT) influences neuronal differentiation capability of induced pluripotent stem cells (iPSC), 3) compare targeting efficiency in neurons and glia, and 4) determine molecular/cellular, behavioral consequences, and pre-clinical therapeutic efficacy of mutant allele-specific CRISPR in vivo. We anticipate this research will 1) nominate optimal CRISPR targeting sites and strategies for HD, 2) generate knowledge base regarding efficiency of mutant-specific CRISPR, and 3) evaluate potential of PAS-based allele-specific CRISPR as therapeutic intervention for HD, providing 1) genetic foundation for novel and innovative therapeutic routes for HD and 2) proof-of-concept for other dominant neurodegenerative diseases.