Fig. 1

Design of two targeting strategies to recover normal splicing/function of the C57BL/6NTac Cdh23 gene. a Design 1 utilises a 121 bp single-stranded oligonucleotide donor (ssODN_U1) in combination with two single guide RNAs (sgRNA_U1 and sgRNA_D1), which flank the Cdh23 ^ahl allele. The full ssODN_U1 donor sequence is shown below the C57BL/6NTac Cdh23 ^ahl sequence (uppercase denotes exonic sequence and lowercase denotes intronic sequence), with homology indicated by dashed lines, the Cdh23 ^ahl allele with an arrow, and two base changes shown in red text. The two synonymous base substitutions are designed to prevent further modification by the CRISPR/Cas9 following repair. The corrected Cdh23 ^753A>G allele is the allele found in inbred mouse strains that do not demonstrate age-related hearing loss (ARHL). The final corrected Cdh23 ^753A>G(U1) gene sequence closely matches that found in these non-ARHL inbred strains at the nucleotide level, except for the two synonymous base substitutions (c.724A > T and c.725G > C; red text). The Cdh23 ^753A>G(U1) product is predicted to be identical to the Cdh23 protein found in non-ARHL mouse strains. b Design 2 also utilises a 121 bp ssODN (ssODN_U2) in combination with two single guide RNAs, sgRNA_D1 also used in design 1 and sgRNA_U2, which lies across the Cdh23 ^ahl locus. The full ssODN_U2 donor sequence is shown below the C57BL/6NTac Cdh23 ^ahl sequence, with homology indicated by dashed lines, the Cdh23 ^ahl allele with an arrow, and one intronic base change shown in red text. The intronic base substitution is designed to prevent further modification by the CRISPR/Cas9 following repair. The final corrected Cdh23 ^753A>G(U2) gene sequence is identical to that found in non-ARHL inbred strains at the nucleotide level, with only an intronic base substitution (c.753 + 9c > t; red text). The Cdh23 ^753A>G(U2) product is predicted to be identical to the Cdh23 protein found in non-ARHL mouse strains