Precise detection of CRISPR-Cas9 editing in hair cells in the treatment of autosomal dominant hearing loss

Chong Cui; Daqi Wang; Bowei Huang; Fang Wang; Yuxin Chen; Jun Lv; Luping Zhang; Lei Han; Dong Liu; Zheng-Yi Chen; Geng-Lin Li; Huawei Li; Yilai Shu

doi:10.1016/j.omtn.2022.07.016

Precise detection of CRISPR-Cas9 editing in hair cells in the treatment of autosomal dominant hearing loss

Mol Ther Nucleic Acids. 2022 Jul 20:29:400-412. doi: 10.1016/j.omtn.2022.07.016. eCollection 2022 Sep 13.

Authors

Chong Cui^{1

2

3}, Daqi Wang^{1

2

3}, Bowei Huang^{1

2

3}, Fang Wang^{1

2

3}, Yuxin Chen^{1

2

3}, Jun Lv^{1

2

3}, Luping Zhang⁴, Lei Han^{1

2

3}, Dong Liu⁵, Zheng-Yi Chen^{6

7}, Geng-Lin Li^{1

3}, Huawei Li^{1

2

3}, 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 Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
³ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai 200031, China.
⁴ Department of Otolaryngology-Head and Neck Surgery, Affiliated Hospital, Nantong University, Nantong 226006, China.
⁵ School of Life Sciences, Nantong Laboratory of Development and Diseases, Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226019, China.
⁶ 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.
⁷ Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114, USA.

Abstract

Gene therapy would benefit from the effective editing of targeted cells with CRISPR-Cas9 tools. However, it is difficult to precisely assess the editing performance in vivo because the tissues contain many non-targeted cells, which is one of the major barriers to clinical translation. Here, in the Atoh1-GFP;Kcnq4 ^+/G229D mice, recapitulating a novel mutation we identified in a hereditary hearing loss pedigree, the high-efficiency editing of CRISPR-Cas9 in hair cells (34.10% on average) was precisely detected by sorting out labeled cells compared with only 1.45% efficiency in the whole cochlear tissue. After injection of the developed AAV_SaCas9-KKH_sgRNA agents, the Kcnq4 ^+/G229D mice showed significantly lower auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) thresholds, shorter ABR wave I latencies, higher ABR wave I amplitudes, increased number of surviving outer hair cells (OHCs), and more hyperpolarized resting membrane potentials of OHCs. These findings provide an innovative approach to accurately assess the underestimated editing efficiency of CRISPR-Cas9 in vivo and offer a promising strategy for the treatment of KCNQ4-related deafness.

Keywords: AAV; CRISPR-Cas9; DFNA2; KCNQ4; MT: RNA/DNA editing; editing efficiency; gene therapy; hair cells; hearing loss.