Arrayed dual-gRNA CRISPR Screens for Excising the C9orf72 Hexanucleotide Repeat Expansion in Patient iPSCs

Hexanucleotide repeat expansion in the first intron of C9orf72 is the leading known genetic cause of both frontotemporal dementia and amyotrophic lateral sclerosis (C9-FTD/ALS). We have previously demonstrated that CRISPR/Cas9 excision of the mutant repeat expansion in patient iPSCs reverts pathological hallmarks of C9-FTD/ALS in iPSC-induced neurons. Here, we aim to identify efficient and safe gRNA targets for CRISPR spCas9 dual-gRNA excision. Utilizing novel ddPCR assays, we screened 120 dual-gRNA pairs, comparing 64 bi-allelic, intronic excisions of the repeat region to 56 allele-specific excisions of the mutant allele in patient iPSCs, ranking them by efficiency. Bi-allelic excisions of the intronic repeat region were more efficient than selective excisions of the mutant allele. Single gRNA indel rates can be averaged to nominate likely efficient dual gRNA pairs, but these pairs must be tested empirically. Neither size of the excision nor the repeat expansion impacted excision efficiency; rather the activity of individual gRNAs drove the excision efficiencies. We found minimal off-targets of the most efficient gRNAs after confirming CHANGE-seq and INDUCE-seq nominated off-targets with whole genome sequencing. This study not only advances the development of targeted therapies for C9orf72-FTD/ALS but establishes a robust framework for selecting efficient and safe CRISPR dual-gRNA pairs in patient iPSCs applicable to many dominant gain-of-function mutations.