Allele-specific gene editing with S. aureus Cas9-KKH prevents deafness in a model of dominant progressive hearing loss

Single nucleotide substitutions that cause dominant hearing loss have been identified in over 30 genes, thus, we explored strategies to specifically and efficiently disrupt a dominant nucleotide substitution in a mouse model of human progressive hearing loss, DFNA36. We focused on the Beethoven mouse, which harbors a dominant substitution in Tmc1 and tested 14 mutation-targeting Cas9/gRNA complexes, including truncated gRNAs, high-fidelity Cas9 enzymes and engineered PAM variants. Only Staphylococcus aureus Cas9 with a PAM recognition variant (SaCas9-KKH) specifically disrupted the dominant Tmc1/TMC1 allele in both mouse and a DFNA36 human cell line, with no detectable disruption of the wild-type (WT) allele. AAV-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to 40 weeks. Viral delivery of gRNAs and SaCas9-KKH with an allele-specific PAM site could potentially be used to disrupt ~21% of alleles that cause dominantly-inherited diseases.