Fig. 4

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