Specific knockdown of Htra2 by CRISPR-CasRx prevents acquired sensorineural hearing loss in mice

Yang Guo; Lei Han; Shuang Han; Honghai Tang; Shengyi Wang; Chong Cui; Bing Chen; Huawei Li; Yilai Shu

doi:10.1016/j.omtn.2022.04.014

Specific knockdown of Htra2 by CRISPR-CasRx prevents acquired sensorineural hearing loss in mice

Mol Ther Nucleic Acids. 2022 Apr 26:28:643-655. doi: 10.1016/j.omtn.2022.04.014. eCollection 2022 Jun 14.

Authors

Yang Guo^{1

2

3}, Lei Han^{1

2

3

4}, Shuang Han^{1

2

3

5}, Honghai Tang^{1

2

3}, Shengyi Wang², Chong Cui^{1

2

3}, Bing Chen^{1

2

3}, Huawei Li^{1

2

3

6}, Yilai Shu^{1

2

3}

Affiliations

¹ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200031, China.
² Institute of Biomedical Science, Fudan University, Shanghai 200032, China.
³ NHC Key Laboratory of Hearing Medicine (Fudan University), Shanghai 200032, China.
⁴ Department of Otorhinolaryngology, The Second Affiliated Hospital, University of South China, Hengyang 421001, China.
⁵ Department of Otolaryngology Head and Neck Surgery, The Second Hospital of Jilin University, Changchun 130041, China.
⁶ The Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China.

Abstract

CasRx, a recently discovered member of the type VI CRISPR system with minimum size, offers a new approach for RNA manipulation with high efficiency and specificity in prokaryotes and eukaryotes. However, in vivo studies of functional recovery using the CasRx system have not been well characterized. Here, we sought to establish an adeno-associated virus (AAV)-CasRx-guide RNA (gRNA) system for the specific knockdown of Htra2 transcript to protect mice from aminoglycosides-induced hearing loss. For the study, we verified an optimized gRNA in vitro, which was packaged into a single AAV with CasRx, and injected the packaged AAV into mice with hearing loss induced by neomycin and auditory functions investigated by auditory brainstem response tests. Upon using the AAV-CasRx-gRNA system, we found the knockdown of Htra2 transcript led to less cochlear hair cell loss and improved auditory function, with low off-target and adverse side effects. Additionally, the decrease in Htra2 significantly inhibits mRNA expression of Casp3 and Casp9. In conclusion, the AAV-CasRx-gRNA-mediated knockdown of Htra2 transcript in mice has been proved effective and safe for preventing hearing loss induced by aminoglycosides and, thus, represents a promising genetic approach for the future clinical applications for treating non-inherited hearing loss.

Keywords: AAV delivery; CRISPR-CasRx; Htra2; RNA editing; acquired sensorineural hearing loss; aminoglycosides.