Preventing autosomal-dominant hearing loss in Bth mice with CRISPR/CasRx-based RNA editing

Ziwen Zheng; Guo Li; Chong Cui; Fang Wang; Xiaohan Wang; Zhijiao Xu; Huiping Guo; Yuxin Chen; Honghai Tang; Daqi Wang; Mingqian Huang; Zheng-Yi Chen; Xingxu Huang; Huawei Li; Geng-Lin Li; Xiaoxiang Hu; Yilai Shu

doi:10.1038/s41392-022-00893-4

Preventing autosomal-dominant hearing loss in Bth mice with CRISPR/CasRx-based RNA editing

Signal Transduct Target Ther. 2022 Mar 14;7(1):79. doi: 10.1038/s41392-022-00893-4.

Authors

Ziwen Zheng^#^{1

2

3}, Guo Li^#^{4

5}, Chong Cui^#^{1

2

3}, Fang Wang^{1

2

3}, Xiaohan Wang⁶, Zhijiao Xu^{1

2

3}, Huiping Guo^{1

2

3}, Yuxin Chen^{1

2

3}, Honghai Tang^{1

2

3}, Daqi Wang^{1

2

3}, Mingqian Huang^{7

8}, Zheng-Yi Chen^{7

8}, Xingxu Huang^{9

10}, Huawei Li^{1

2

3

11}, Geng-Lin Li^{12

13

14}, Xiaoxiang Hu^{15

16}, Yilai Shu^{17

18

19}

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 Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
³ NHC Key Laboratory of Hearing Medicine (Fudan University), Shanghai, 200032, China.
⁴ State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, 100193, China.
⁵ College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
⁶ School of Life Science and Technology, Southeast University, Nanjing, 210096, China.
⁷ Department of Otolaryngology-Head and Neck Surgery, Graduate Program in Speech and Hearing Bioscience and Technology and Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA.
⁸ Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA, 02114, USA.
⁹ School of Life Science and Technology, ShanghaiTech University, Shanghai, 200031, China.
¹⁰ CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China.
¹¹ Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, 200031, China.
¹² 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. genglin.li@fdeent.org.
¹³ Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China. genglin.li@fdeent.org.
¹⁴ NHC Key Laboratory of Hearing Medicine (Fudan University), Shanghai, 200032, China. genglin.li@fdeent.org.
¹⁵ State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, 100193, China. huxx@cau.edu.cn.
¹⁶ College of Biological Sciences, China Agricultural University, Beijing, 100193, China. huxx@cau.edu.cn.
¹⁷ 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. yilai_shu@fudan.edu.cn.
¹⁸ Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China. yilai_shu@fudan.edu.cn.
¹⁹ NHC Key Laboratory of Hearing Medicine (Fudan University), Shanghai, 200032, China. yilai_shu@fudan.edu.cn.

^# Contributed equally.

Abstract

CRISPR/RfxCas13d (CasRx) editing system can specifically and precisely cleave single-strand RNAs, which is a promising treatment for various disorders by downregulation of related gene expression. Here, we tested this RNA-editing approach on Beethoven (Bth) mice, an animal model for human DFNA36 due to a point mutation in Tmc1. We first screened 30 sgRNAs in cell cultures and found that CasRx with sgRNA3 reduced the Tmc1^Bth transcript by 90.8%, and the Tmc1 wild type transcript (Tmc1⁺) by 44.3%. We then injected a newly developed AAV vector (AAV-PHP.eB) based CasRx into the inner ears of neonatal Bth mice, and we found that Tmc1^Bth was reduced by 70.2% in 2 weeks with few off-target effects in the whole transcriptome. Consistently, we found improved hair cell survival, rescued hair bundle degeneration, and reduced mechanoelectrical transduction current. Importantly, the hearing performance, measured in both ABR and DPOAE thresholds, was improved significantly in all ages over 8 weeks. We, therefore, have validated the CRISPR/CasRx-based RNA editing strategy in treating autosomal-dominant hearing loss, paving way for its further application in many other hereditary diseases in hearing and beyond.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Clustered Regularly Interspaced Short Palindromic Repeats
Hearing Loss* / genetics
Hearing Loss, Sensorineural* / genetics
Membrane Proteins / genetics
Mice
RNA Editing

Substances

Membrane Proteins
TMC1 protein, mouse