Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.