Adeno-associated virus vector enables safe and efficient Cas9 activation in neonatal and adult Cas9 knockin murine cochleae.

Adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene manipulation in the inner ear. For example, hair cells (HCs) and multiple other cell types can be transduced by the local injection of AAVs into the inner ear. However, application of the AAV-mediated CRISPR/Cas9 gene-editing approach to the inner ear in adult mice has not yet been studied. Based on our previous work, we investigated several AAV serotypes in neonatal and adult mice in parallel, and found that AAV8 had the top efficiency to transduce inner HCs. We then tested the ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knockin mice, and observed significant Cas9 activation in the inner ear of both neonatal and adult animals. Neither the AAV8 virus itself nor the surgical procedures used to deliver it-cochleostomy for neonatal mice and canalostomy for adult mice-caused any damage to HCs or impaired normal hearing. Our studies indicate that the local injection of AAV8-Cre can induce Cas9 activation to perform safe and efficient gene editing in the inner ear, expanding the repertoire of gene-editing tools for regulating gene expression in the inner ear as a part of efforts to rescue genetic hearing loss, initiate regeneration of HCs, or develop gene therapy techniques.