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

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.

Fig. 1

Screening for efficient and specific sgRNAs for targeting the Tmc1^Bth transcript. a Constructs used for the sgRNA screen mediated by the Cas RNA editing system. Five vectors were constructed, including the Cas expression vector, the mCherry-Tmc1^Bth fluorescence reporter, the mCherry-Tmc1⁺ fluorescence reporter, the sgRNA expression vectors for targeting the Tmc1^Bth transcript, and the non-targeting (NT) sgRNA expression vector. b Ratios of fluorescence intensity with sgRNAs compared to control sgRNA (NT) for targeting mCherry-Tmc1^Bth mRNA and mCherry-Tmc1⁺ mRNA mediated by CasRx system. Data are shown as the mean ± SD (n = 3 biologically independent samples). c Ratios of fluorescence intensity with sgRNAs compared to control sgRNA (NT) for targeting mCherry-Tmc1^Bth mRNA and mCherry-Tmc1⁺ mRNA mediated by PspCas13b system. Data are shown as the mean ± SD (n = 3 biologically independent samples). d Mean ratio of fluorescence intensities between mCherry-Tmc1^Bth and mCherry-Tmc1⁺ mRNA. sgRNA3 has the lowest mean ratio of fluorescence intensity for all the 30 sgRNAs tested. e The integrated fluorescence intensity of cells with CasRx system. The integrated fluorescence density was significantly decreased with sgRNA3 targeting mCherry-Tmc1^Bth mRNA compared to targeting mCherry-Tmc1⁺ mRNA, and integrated fluorescence density was decreased comparted to targeting mCherry-Tmc1^Bth and mCherry-Tmc1⁺ mRNA with non-targeting sgRNA. Data are shown as the mean ± SD (n = 5 biologically independent samples). ***p < 0.001, P-values were determined by one-way ANOVA with Sidak’s multiple comparisons test

Fig. 2

Off-target analysis for RNA editing in 293T cells by RNA-Seq. a Off-target-1 to Off-target-10 are ten off-target sites detected by RNA-seq. Mismatches compared to the on-target site are shown and highlighted in color. The 30 bp sequence (On-target) targeted by the sgRNA3 is shown in the top row. b Off-target analysis for RNA editing in 293T cells by RNA-Seq. No significant difference was found in CasRx + sgRNA3 + mCherry-Tmc1^Bth, CasRx + sgRNA3 + mCherry-Tmc1⁺ or sgRNA3, when compared to EGFP. Data are shown as the mean ± SD. ns no significance. Statistical analysis was performed by multiple unpaired t-test

Fig. 3

CasRx selectively disrupts the Tmc1^Bth transcript in Bth mice. a Schematic of the AAV vector encoding CasRx and sgRNA3 (upper), and a control NT vector (lower). b Outline of the in vivo experiments. Mice were injected with AAV (~5 × 10⁹ vg) at P1–P2, and the organs of Corti were dissected and cultured at P5, and hair cell physiology was analyzed at P15–P16. Injected mice were sequenced after 2 weeks followed by hearing tests (ABR and DPOAE) after 4, 8, and 12 weeks, immunohistochemistry, and scanning electron microscopy at 10 weeks after injection. c The percentage of deep sequencing reads of Tmc1^Bth and Tmc1⁺. Pie charts indicate the mean composition of Tmc1^Bth and Tmc1⁺ transcripts in these samples, sequences show the single-nucleotide difference between the Tmc1^Bth and Tmc1⁺ transcripts (52.83 ± 5.33%, 53.39 ± 4.8%, and 14.88 ± 9.77% Tmc1^Bth transcript for non-injected, injected with AAV-CasRx + NT, and injected with AAV-CasRx + sgRNA3, respectively. n = 3 mice, data are shown as the mean ± SD). d Deep sequencing analysis of the ratios of transcripts between Tmc1^Bth and Tmc1⁺ for non-injected mice (n = 3 mice), mice injected with AAV-CasRx + NT (n = 3 mice), and mice injected with AAV-CasRx + sgRNA3 (n = 3 mice), respectively. Data are shown as the mean ± SD, **p < 0.01, P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. e mRNA expressions in the cochlea at 2 weeks after injection as measured by RT-qPCR. The expression of CasRx mRNA (n = 11 mice), total Tmc1 mRNA (n = 5 mice), Tmc1^Bth mRNA (n = 4 mice), and Tmc1^Bth (n = 5 mice) between injected with AAV-CasRx + sgRNA3 and non-injected contralateral ears were showed in graphs. Relative mRNA expression levels were calculated with the ΔΔCt algorithm. Data are shown as the mean ± SD, *p < 0.05, ***p < 0.001, P-value was determined by unpaired two-tailed t-test. f Amplification of the Tmc1^Bth sequence. The amplicon was detected by a pair of specific targeting primers with heterozygous templates, and the primers cannot amplify with the wild-type template. g Representative MET recordings and maximal MET current amplitudes of apical IHCs at the equivalent of P15–P16. h The MET current amplitude was 461.134 ± 74.978 pA, 442.458 ± 82.805 pA, and 344.409 ± 114.591 pA in Tmc^+/+ mice (n = 16 OHCs), non-injected Tmc1^Bth/Bth mice (n = 18 OHCs), and Tmc1^Bth/Bth mice injected with AAV-CasRx + sgRNA3 (n = 16 OHCs), respectively. Data are shown as the mean ± SD, **p < 0.01, P-values were determined by one-way ANOVA with Sidak’s multiple comparisons test

Fig. 4

Improvement in auditory function via CasRx in Bth mice. a ABR waveforms recorded at 8 kHz at 4 weeks in the Tmc1^Bth/+ injected right ear, the non-injected left ear, and the wild-type ear. The green traces indicate the threshold. b, c Tone-burst and click-evoked ABR thresholds at 4, 8 weeks in Tmc1^+/+ (Green), Tmc1^Bth/+ + AAV-CasRx + sgRNA3 (Blue), and Tmc1^Bth/+ non-injected contralateral (Red) ears. Mean ABR thresholds were significantly reduced in ears injected with AAV-CasRx + sgRNA3 (~5 × 10⁹ vg of AAV) compared to non-injected Tmc1^Bth/+ ears after 4 and 8 weeks. Statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for multiple comparisons. d DPOAE thresholds at 4, 8 weeks in Tmc1^Bth/+ + AAV-CasRx + sgRNA3 (Blue) and Tmc1^Bth/+ untreated contralateral (Red) ears. DPOAE thresholds were significantly reduced in the ears injected with AAV-CasRx + sgRNA3 (~5 × 10⁹ vg of AAV) at two frequencies. Statistical analysis was performed by two-way Bonferroni’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Values and error bars represent the mean ± SD

Fig. 5

Injection of AAV-CasRx + sgRNA3 (~5 × 10⁹ vg of AAV) protects hair cells and hair bundles. a Representative confocal images of 100 μm cochlear sections harvested 10 weeks after injection. Samples were stained with myosin7a (Red). The images are from Tmc1^+/+, Tmc1^Bth/+ + AAV-CasRx + sgRNA3, and Tmc1^Bth/+ non-injected mice (n = 5 mice) at locations corresponding to 8 and 16 kHz. The IHCs and OHCs are indicated. Scale bar: 20 μm. b The number of OHCs (upper) and IHCs (lower) per 100 μm of the cochleae. Data are shown as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical analysis was performed by two-way Sidak’s multiple comparisons test. c SEM images of the apical cochlear sensory epithelium showing the morphology of the hair cell bundles. Tmc1^+/+, Tmc1^Bth/+ + AAV-CasRx + sgRNA3, and Tmc1^Bth/+ non-injected samples were collected 10 weeks after injection. Scale bars: 20 μm (upper); 3 μm (lower)

Fig. 6

Off-target analysis for RNA editing in vivo by RNA-Seq. a Off-target-1 to Off-target-10 were ten off-target sites detected by RNA-seq. Mismatches compared to the on-target site are shown and highlighted in color. The 30 bp sequence (On-target) targeted by the sgRNA3 is shown in the top row. b Comparison of FPKM values of the ten off-target sites at injected AAV-CasRx + sgRNA3 or non-injected ears. Data are shown as the mean ± SD. ns no significance. Statistical analysis was performed by multiple unpaired t-test