Extended Data Fig. 6: Generation and characterization of Abca4^hu1961E mice.

A) Strategy for the generation of the Abca4^hu1961E mouse line. Abca4 exon 42 (blue box) with flanking introns. The PAM sites are highlighted, and the gRNA binding sites are indicated by black dashed lines. The red arrowhead points to the p.Gly1961Glu mutation. Bold nucleotides indicate nucleotide changes due to humanization. Note the deletion in the downstream intron – this is intentional and was introduced to disrupt the PAM site. The deletion is not expected to interfere with splicing as it is at position +9, at which there is no base preference for canonical splicing⁵². B) Sequencing of the gDNA of the Abca4^hu1961E allele. The red arrowhead points to the Abca4 c.5882G>A mutation, the black arrowhead points to a deletion in the intron. C) Deep-sequencing of Abca4^{hu1961E/ms1961G(KO)} mice, where the results indicate heterozygosity. The red arrowhead points to the Abca4 c.5882G>A mutation and the black arrowheads point to the nucleotide changes due to humanization. D) Retinoid and bisretinoid levels measured by LC-HRMS analysis of eyes of Abca4^{hu1961E/ms1961G(KO)} mice compared to age-matched Abca4^{ms1961G/ms1961G(KO)} littermates. Retinyl acetate was used as internal standard (IS) for normalization. Absolute levels of A2E (A2E targeted) were determined according to the standard curve for synthetic A2E. Results were obtained from four biological replicates, except for atROL for mutant animals, where 3 biological replicates were used (9 months). Results were obtained from six biological replicates, except for atROL, where 4 biological replicates were used (11 months). Results are presented as mean ± s.d. Statistical tests used were two-sided, A2E targeted: *P = 0.021, A2GPE: *P = 0.016, dimeric atRAL: **P = 0.002, atROL: P = 0.056 for the interaction between genotype and age in the ANOVA model. E) Fundus autofluorescence images of retinas from an Abca4^{hu1961E/ms1961G(KO)} and wild-type Abca4^ms1961G/G mouse (top). Quantification of the fluorescent signals at different ages (bottom). For Abca4^{hu1961E/ms1961G(KO)} animals, results were obtained from eight (week 20), nine (week 33) and five (week 44) biological replicates (eyes). For wild-type Abca4^ms1961G/G animals, results were obtained from eight (week 20), two (week 33) and five (week 44) biological replicates (eyes). F) Confocal images of sections from Abca4^{hu1961E/ms1961G(KO)} mice. Grey: Hoechst; cyan: rhodopsin, magenta: ABCA4 (scale bar: 25 µm).