Fig 2: Targeting the Htra2 gene with the SaCas9 system. a Design of the gRNAs. The three gRNA targeting sequences of the Htra2 gene are listed. PAM sites are marked by red nucleotides. The red arrows indicate the exons targeted by gRNAs. The gray arrows indicate the primers. b Experimental overview of the in vivo studies of the Anc80L65–SaCas9 therapeutic system. c The indel frequency of the gRNAs was determined in HEI-OC1 cells by high-throughput sequencing. Dots represent individual values and bars represent the mean ± SD, n = 2. d The indel frequency of the gRNA (Sa-g3) from the injected and non-injected ears in vivo. Dots represent individual values and bars represent the mean ± SD, n = 4. e The indels causing in-frame versus frameshift mutations (percentages are shown) in the coding sequence after the plasmid of SaCas9–gRNA (Sa-g3) transfection in HEI-OC1 cells in vitro. f Indels causing in-frame versus frameshift mutations in the coding sequence after Anc80L65–SaCas9–Htra2 gRNA injection in vivo. g Indel profiles from the cochleae injected with Anc80L65–SaCas9–Htra2 gRNA in vivo. Negative numbers represent deletions and positive numbers represent insertions. h The top 10 indel mutations detected by deep sequencing after Anc80L65–SaCas9–Htra2 gRNA injection in vivo

Fig 3: In vitro genome editing with the SpCas9 therapeutic system. a Design of the gRNAs. The three gRNA targeting sequences in the Htra2 gene are listed. PAM sites are marked by red nucleotides. The red arrows indicate the exons targeted by the gRNAs, and the gray arrows indicate the primers. b The indel frequency of the gRNAs was tested in HEI-OC1 cells using high-throughput sequencing. Dots represent individual values and bars represent the mean ± SD, n = 2. c Indels causing in-frame versus frameshift mutations (percentages are shown) in the coding sequence after transfection with SpCas9–gRNA. d Indel profiles from HEI-OC1 cells transfected with Sp-g1, Sp-g2, and Sp-g3, respectively. Negative numbers represent deletions, and positive numbers represent insertions. nt nucleotides

Fig 4: The Anc80L65–SpCas9–Htra2 gRNA therapeutic system protected hair cells from neomycin-induced injury in vitro. a Experimental procedures for the in vitro studies with application of the Anc80L65–SpCas9 therapeutic system. b The indel frequency of the gRNAs was measured in the cultured basilar membrane at 8 days post-transduction with the Anc80L65–SpCas9 therapeutic system using high-throughput sequencing. Dots represent individual values, and bars represent the mean ± SD, n = 3. c Indels causing in-frame versus frameshift mutations in the coding sequence after co-transduction of Anc80L65–SpCas9 and Anc80L65–gRNA. d Representative confocal images of neomycin-treated cochlear explants without Anc80L65–SpCas9–Htra2 gRNA treatment. e Representative confocal images of Myo7a and caspase-3 immunofluorescence in neomycin-treated cochlear explants treated with Anc80L65–SpCas9–Htra2 gRNA for 8 days prior to neomycin exposure. IHC inner hair cell, OHC outer hair cell. Both scale bars (in d and e) represent 20 µm long and apply to all images. f–h Comparison of the numbers of Myo7a+ hair cells (f), caspase-3+ cells (g), and Myo7a+/caspase-3+ hair cells (h) per 100-µm sensory epithelium region between the neomycin-treated groups with or without Anc80L65–SpCas9–Htra2 gRNA treatment in the apical, middle, and basal turns of the cochlea. i Comparison of the ratio of Myo7a+/caspase-3+ cells to Myo7a+ cells between the neomycin-treated groups with or without Anc80L65–SpCas9–Htra2 gRNA treatment. Dots represent individual values, and bars represent the mean ± SD in f–i. For statistical analysis in f–i, Mann–Whitney U-tests, unpaired two-tailed Student’s t-tests, or unpaired t-tests with Welch’s correction were used as applicable. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5

Fig 5: Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells.MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, NutramilTM Complex, at 4% concentration; NC-CC, NutramilTM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 µM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). ß-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).