Massively Parallel Splicing Assay to Examine Splicing Errors Caused by Disease-Related Intronic Variants

Splicing errors represent 10-30% of the pathogenic mutations responsible for rare genetic disorders. RNA splicing ensures proper gene expression by selectively joining exons and removing introns, with key regulatory sequences being located within the introns. The 5' splice site and branch site interact with small nuclear RNAs to form the spliceosome's recognition complex, while elements such as the polypyrimidine tract and splicing enhancers/silencers recruit proteins to regulate spliceosome assembly. Predicting splicing disruptions from intronic variants is challenging due to the complexity of these interactions. Intronic variants, comprising 90% of natural human gene variations, may disrupt canonical splicing and give rise to disease. To investigate this possibility, we developed a massively parallel splicing assay (MaPSy) to assess patient-identified intronic variants. Synthesized oligonucleotides with reference or variant sequences were ligated into splicing minigenes containing promoter and polyadenylation signals. Each construct included two constant exons flanking a middle exon that harbored the variable intron-exon junction sequence of interest. The cellular splicing efficiency of the variant sequences was compared to reference counterparts, allowing us to identify significant disruptions as splicing variants. The results of the MaPSy can be validated through additional approaches, such as minigene assays or CRISPR-mediated genome editing in vivo. Furthermore, aggregate analysis of the disrupted junctions can provide deeper insights into splicing mechanisms and the molecular basis of diseases associated with splicing errors.