Research ArticleGENE EDITING

In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness

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Science Translational Medicine  03 Jun 2020:
Vol. 12, Issue 546, eaay9101
DOI: 10.1126/scitranslmed.aay9101
  • Fig. 1 Evaluating different base editor and guide RNA combinations to correct the Tmc1Y182C/Y182C allele in Baringo MEFs.

    (A) Schematic of the Tmc1 locus highlighting the c.A545G mutation (red), silent bystander bases (purple), and three candidate guide RNAs that position the target C (in red) at different protospacer positions (C8, C7, and C10) and use different PAMs (AGG, GGA, and TGA, in blue). (B) Base editing efficiencies for the four CBE-P2A-GFP variants tested with sgRNA1 (where the four CBEs are APOBEC1-BE4max, CDA1-BE4max, evoCDA1-BE4max, or AID-BE4max). Base editing values (blue bars) reflect correction of the Baringo mutation to the wild-type TMC1 protein coding sequence, with no other nonsilent changes or indels. Three days after nucleofection into Baringo MEFs, GFP-positive (GFP+) cells were sorted, and genomic DNA was characterized by HTS. (C) Base editing efficiencies for three different guide RNAs tested with AID-BE4max variants: AID-BE4max + sgRNA1, AID-VRQR-BE4max + sgRNA2, or AID-VRQR-BE4max + sgRNA3. Three days after nucleofection of these plasmids into Baringo MEFs, GFP-positive cells were sorted and sequenced by HTS. (D) Base editing efficiencies in Baringo MEFs after a 14-day incubation with dual AAV encoding AID-BE3.9max + sgRNA1 at high (N terminus, 6.1 × 108 vg; C terminus, 8.3 × 108 vg) and low (N terminus, 3.1 × 107 vg; C terminus, 4.2 × 107 vg) doses. Dots, colored bars, and error bars represent individual biological replicates, mean values, and SEM, respectively (n = 3 to 5).

  • Fig. 2 In vivo base editing of Tmc1Y182C/Y182C in Baringo mice, in vitro off-target analysis for sgRNA1, and in vivo analysis of hair cell stereocilia bundle morphology.

    (A) The 10 most abundant genomic DNA cleavage products (which include the on-target site and nine potential off-target sequences) from Cas9 nuclease + sgRNA1 as identified in vitro by CIRCLE-seq, aligned to the on-target Tmc1 sequence. (B) Editing analysis of the nine candidate off-target sites identified by CIRCLE-seq in MEFs treated with dual AAV encoding AID-BE3.9max + sgRNA1. The on-target locus, plus the top nine off-target sites identified by CIRCLE-seq, was sequenced by HTS. Dots and bars represent biological replicates and mean ± SEM (n = 3). (C) Efficiency of AID-BE3.9max + sgRNA1–mediated editing in treated Baringo (Tmc1Y182C/Y182C;Tmc2+/+) mice. Mouse inner ears were injected at P1 with 1 μl (3.1 × 109 vg of each AAV) of dual AAV encoding AID-BE3.9max + sgRNA1. After 14 days, cochleae were microdissected into base, mid, and apex samples. Genomic DNA was extracted from each sample and sequenced by HTS. Each dot represents the efficiency of generating Tmc1 alleles with wild-type TMC1 protein sequence and no other nonsilent mutations or indels, averaging all samples sequenced from one injected cochlea. To obtain Tmc1 mRNA from the cochlea, we extracted the cochlea at P30, isolated RNA, reverse transcribed into cDNA, and analyzed by HTS. Each dot represents the mRNA from one injected cochlea. (D to F) Representative SEM images at the apical turn of OHCs and IHCs of (D) wild-type (Tmc1+/+;Tmc2+/+) mice, (E) untreated Baringo (Tmc1Y182C/Y182C;Tmc2+/+) mice, and (F) Baringo mice treated with dual AAV encoding AID-BE3.9max + sgRNA1. The organs of Corti were imaged by SEM at 4 weeks. Scale bar, 10 μm.

  • Fig. 3 Inner-ear injection of dual AAV encoding AID-BE3.9max + sgRNA1 restores sensory transduction in Tmc1Y182C/Y182C;Tmc2∆/∆ IHCs.

    (A) Confocal images of midturn cochlear sections excised from P5 Tmc1Y182C/Y182C;Tmc2∆/∆ mouse cochleae. A representative untreated mouse (top panel) or a representative mouse treated with 1 μl (3.1 × 109 vg of each AAV) of dual AAV encoding AID-BE3.9max + sgRNA1 (bottom panel) are shown. The tissue was cultured for 9 to 13 days and treated with 5 μM FM1-43 for 10 s followed by three full bath exchanges to wash out excess dye. The tissue was mounted and imaged for FM1-43 uptake (green) in IHCs and OHCs. All images are 500 × 100 μm. Scale bars, 50 μm. (B) Quantification of FM1-43–positive IHCs (P = 0.0072) and OHCs (P = 0.026) from untreated (gray) and treated (purple) mice represented as mean ± SD. (n = 3 to 4 different mice in each group). (C) Representative families of sensory transduction currents evoked by mechanical displacement of hair bundles recorded from apical IHCs of untreated Tmc1Y182C/Y182C;Tmc2∆/∆ mice at P8 (untreated, orange), from Tmc1Y182C/Y182C;Tmc2∆/∆ mice treated with dual AAV encoding AID-BE3.9max + sgRNA1 at P14 (blue) and P18 (purple) and from wild-type (WT) Tmc1+/+;Tmc2 +/+ mice at P14 to P16 (gray). Horizontal lines and error bars reflect mean values and SD of three to four independent mice and four to eight hair cells (indicated on top of x axis), with each dot representing one IHC.

  • Fig. 4 Dual AAV base editor treatment partially restores auditory function in Baringo (Tmc1Y182C/Y182C;Tmc2+/+) mice.

    (A) Representative sets of ABR waveforms recorded in response to 5.6-kHz tone bursts of varying sound intensity for untreated Baringo mice (left) and Baringo mice treated with dual AAV encoding AID-BE3.9max + sgRNA1 (right). (B) Same as (A), but with untreated wild-type mice (left) and wild-type mice treated with 1 μl (3.1 × 109 vg of each AAV) of dual AAV encoding AID-BE3.9max + sgRNA1 (right). (C) Mean ABR responses for all four groups (untreated and treated, Baringo and wild-type mice) across all tested frequencies. Untreated Baringo mice (black, n = 10) are shown with no detectable ABR threshold (>110 dB, indicated by the upward arrows). Among the treated Baringo mice (n = 15) injected with dual AAV encoding AID-BE3.9max + sgRNA1, nine showed ABR response improvements of up to >50 dB indicated with purple lines, while six did not show any ABR threshold changes shown with gray lines. Untreated wild-type mice in blue (n = 6). Wild-type mice injected with dual AAV encoding AID-BE3.9max + sgRNA1 in orange (n = 4). (D) The same mice in (C) were subjected to DPOAE testing. All recordings were done at mice age of 4 weeks (P30). (E) Mean ABR responses for three animals tested at two time points (4 and 6 weeks) across all tested frequencies. Baringo mice (n = 3) injected with dual AAV encoding AID-BE3.9max + sgRNA1 at P1 and tested at 4 weeks showed (up to 65 dB) hearing improvement (yellow, blue, and purple solid lines). The same animals were retested at 6 weeks (dashed lines). Solid and dashed lines of the same color denote the same animal tested at 4 and 6 weeks, respectively. Values and error bars reflect mean ± SEM for the numbers of mice specified above.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/546/eaay9101/DC1

    Methods

    Fig. S1. Base editing outcomes from different CBE and sgRNA combinations.

    Fig. S2. Anc80-Cbh-GFP AAV transduction in IHCs and OHCs in wild-type mice.

    Fig. S3. Base editing outcomes from mRNA of treated and untreated Baringo cochlea.

    Fig. S4. Base editing at on-target and off-target genomic DNA sites identified by CIRCLE-seq using Cas9 + sgRNA1.

    Fig. S5. Transduction currents from IHCs and OHCs of Tmc1Y182C/Y182C;Tmc2+/+ and Tmc1Y182C/Y182C;Tmc2/ mice at different time points.

    Fig. S6. Hair cell morphology in the organ of Corti from Tmc1Y182C/Y182C;Tmc2+/+ mice with and without treatment with dual AAV-AID-BE3.9max + sgRNA1.

    Fig. S7. Hair bundle morphology in the basal turn of the organ of Corti from Tmc1Y182C/Y182C;Tmc2+/+ mice with and without treatment with dual AAV-AID-BE3.9max + sgRNA1.

    Fig. S8. Auditory function and correlation of ABR thresholds with surviving hair cells in Baringo mice at 4 and 6 weeks.

    Table S1. P value calculation on ABR and DPOAE data from untreated wild-type mice and wild-type mice treated with dual AAV encoding AID-BE3.9max + sgRNA1.

    Table S2. P value calculation on ABR data (5.6 to 16 kHz) from Baringo mice treated with dual AAV encoding AID-BE3.9max + sgRNA1 that showed ABR responses and untreated Baringo mice.

    Table S3. Primers used for HTS.

    Table S4. CRISPResso2 output for base editing at the target locus.

    Table S5. List of base editing targets to correct known pathogenic point mutations in TMC1.

    Data file S1. Single data points and exact P values.

    References (67, 68)

  • The PDF file includes:

    • Methods
    • Fig. S1. Base editing outcomes from different CBE and sgRNA combinations.
    • Fig. S2. Anc80-Cbh-GFP AAV transduction in IHCs and OHCs in wild-type mice.
    • Fig. S3. Base editing outcomes from mRNA of treated and untreated Baringo cochlea.
    • Fig. S4. Base editing at on-target and off-target genomic DNA sites identified by CIRCLE-seq using Cas9 + sgRNA1.
    • Fig. S5. Transduction currents from IHCs and OHCs of Tmc1Y182C/Y182C;Tmc2+/+ and Tmc1Y182C/Y182C;Tmc2/ mice at different time points.
    • Fig. S6. Hair cell morphology in the organ of Corti from Tmc1Y182C/Y182C;Tmc2+/+ mice with and without treatment with dual AAV-AID-BE3.9max + sgRNA1.
    • Fig. S7. Hair bundle morphology in the basal turn of the organ of Corti from Tmc1Y182C/Y182C;Tmc2+/+ mice with and without treatment with dual AAV-AID-BE3.9max + sgRNA1.
    • Fig. S8. Auditory function and correlation of ABR thresholds with surviving hair cells in Baringo mice at 4 and 6 weeks.
    • Table S1. P value calculation on ABR and DPOAE data from untreated wild-type mice and wild-type mice treated with dual AAV encoding AID-BE3.9max + sgRNA1.
    • Table S2. P value calculation on ABR data (5.6 to 16 kHz) from Baringo mice treated with dual AAV encoding AID-BE3.9max + sgRNA1 that showed ABR responses and untreated Baringo mice.
    • Table S3. Primers used for HTS.
    • Table S4. CRISPResso2 output for base editing at the target locus.
    • Table S5. List of base editing targets to correct known pathogenic point mutations in TMC1.
    • References (67, 68)

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Single data points and exact P values.

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