Most forms of hereditary deafness are recessive, but some show a dominant mode of inheritance. If the dominant allele could be disrupted, leaving a single normal allele, it might prevent the disease. As a model for human dominant deafness, we used Beethoven (Bth) mice, which harbor a point mutation in the Tmc1 gene encoding the hair-cell transduction channel. In a collaboration with the laboratories of Jeff Holt and Gwen Géléoc and of Keith Joung, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. Single nucleotide discrimination can be difficult because Cas9 enzymes can tolerate mismatches between guide RNA and target DNA. The protospacer-adjacent motif (PAM) in some Cas9 enzymes can also tolerate mismatches. To circumvent these limitations, we identified a variant of Cas9 (SaCas9-KKH) that can uniquely distinguish the pathologic mutation using its PAM site, and that selectively and efficiently disrupted the mutant but not the wild-type Tmc1 allele in Bth mice. This strategy prevented deafness in Bth mice up to one year. More than a fifth of all dominant human mutations could be targeted using a similar approach.

Rescue of hair bundle morphology in apical cochlear sensory epithelium of Beethoven mice. Top, wild-type mouse.  Middle, heterozygous Bth mice. Hair bundles are severely disorganized. Bottom, heterozygous Bth mice treated with AAV encoding SaCas9-KKH and specific gRNA. Most hair cells survive and have well-shaped bundles. (image: Carl Nist-Lund)

PAPERS

György B, Nist-Lund C, Pan B, Asai Y, Karavitaki KD, Kleinstiver BP, Garcia SP, Zaborowski MP, Solanes P, Spataro S, Schneider BL, Joung JK, Géléoc GSG, Holt JR, Corey DP. (2019) Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss. Nat Med. 25:1123-1130