“We developed a better, as more precise version for CRISPR-Cas9 gene-editing to prevent the hearing loss. It allows us to selectively shut down the defective copy of the gene Tmc1, which is the disease-causing mutation for the hearing loss in Beethoven mice. The healthy copy of the gene remains unaffected. This is really remarkable, because we are talking about a single incorrect DNA letter in the defective Tmc1 copy, in a score of three billion letters in the mouse genome.”said study first author Bence Gyorgy, who conducted the work while at Harvard Medical School and is now at the Institute of Molecular and Clinical Ophthalmology in Basel, Switzerland.
Since most dominant human mutations are single nucleotide substitutions, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.
Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss
Bence György, Carl Nist-Lund, Bifeng Pan, Yukako Asai, K. Domenica Karavitaki, Benjamin P. Kleinstiver, Sara P. Garcia, Mikołaj P. Zaborowski, Paola Solanes, Sofia Spataro, Bernard L. Schneider, J. Keith Joung, Gwenaëlle S. G. Géléoc, Jeffrey R. Holt & David P. Corey | Nature Medicine (2019) | published online 03 July 2019