Fig. 4: SPLICER Skips APP Exon 17 in vitro and Reduces Production of Aβ42.

A Schematic representation of APP and sequence of APP exon 17 and introns 16 and 17. B Genomic DNA editing rates at the SA of APP exon 17 with ABE8e and CBE4max fused with SpCas9 or SpRY Cas9 in HEK293T cells. C SA DNA editing rates with SpRY Cas9 ABE8e when editing the SA alone or in combination with a panel of sgRNAs targeting the SD. D SD DNA editing rates with sgRNAs targeting the SD alone or in combination with a sgRNA targeting the SA. Editing at the canonical and cryptic SDs is highlighted. E APP exon 17 exon skipping rates with an sgRNA targeting the SA in combination with one of 3 different sgRNAs targeting the SD measured by NGS. F qPCR quantification of total APP mRNA. G, H Quantification of APP exon 17 exon splicing (top) and schematic representation of the corresponding splicing event (bottom) following targeting of the SA with a sgRNA and SD with two different sgRNAs. (I) DNA editing at the SA and (J) SD of APP exon 17 in BE(2)-M17 neuroblastoma cells following enrichment with puromycin. K Exon skipping rates for APP exon 17 in puromycin selected BE(2)-M17 neuroblastoma cells. L Aβ42 levels in BE(2)-M17 cells following skipping of APP exon 17 in comparison with control cells using ELISA. All measurements of full-length and cryptic exon skipping were performed by NGS while all DNA editing rates were measured via Sanger sequencing. For all bar graphs, values represent means and error bars indicate SD. For heatmaps, values represent means. For sashimi plots, values represent means. In experiments performed in HEK293 cells, each replicate is a biological replicate originating from an independent transfection of each sgRNA. In experiments with BE(2)-M17 cells, a single transfection was performed with each sgRNA set to generate a puromycin selected cell line. Following cell line generation, each sgRNA set was maintained amongst three separate plates with each replicate deriving from one plate; n = 3 for all experiments; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA, Tukey’s post hoc comparing SA/SD to SA and SD except for (B, L), which were analyzed via a two-tailed, unpaired t-test. Source data are provided as a Source Data file.