CRISPR gene editing holds promise to cure or arrest genetic disease, if we can find and implement curative edits reliably, safely and effectively. Expansion of a hexanucleotide repeat in C9orf72 is the leading known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We evaluated three approaches to editing the mutant C9orf72 gene for their ability to correct pathology in neurons derived from patient iPSCs: excision of the repeat region, excision of the mutant allele, and excision of regulatory region exon 1A. All three approaches normalized RNA abnormalities and TDP-43 pathology, but only repeat excision and mutant allele excision completely eliminated pathologic dipeptide repeats. Our work sheds light on the complex regulation of the C9orf72 gene and suggests that because of sense and anti-sense transcription, silencing a single regulatory region may not reverse all pathology. Our work also provides a roadmap for evaluating CRISPR gene correction using patient iPSCs.

This protocol describes how to conjugate antibodies and run the Meso Scale Discovery (MSD) Sandwich enzyme-linked immunosorbent assay (ELISA) on MSD GOLD 96-well Small Spot Streptavidin SECTOR Plates. This protocol is adapted from MSD GOLD™ Streptavidin Plate and Avidin Plates Quick Guide 1 and MSD GOLD™ SULFO-TAG NHS-Ester Conjugation Quick Guide 2 and optimized for C9orf72 dipeptide repeat detection from human iPSC derived neurons.

This protocol describes how to perform gene editing on human induced pluripotent stem cells (iPSCs) via ribonucleoprotein (RNP) and how to the isolate lines with the desired excision. It describes nucleofection, single cell sorting via FACS, genotyping, and the maintenance of the cell lines throughout the process. This protocol is optimized for spCas9.