Abstract
Pre-mRNA splicing is an important mechanism by which genetic variation influences complex traits. We developed a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS) that allows us to qantify exon inclusion in large libraries of human exons and surrounding intronic contexts. We used MFASS to explore >10,000 designed mutations intended to alter regulatory elements that govern splicing. Many classes of mutations led to large-effect splicing disruptions including mutations far from canonical splice sites, and these effects were not easily predicted. We assayed 29,531 extant variants in the Exome Aggregation Consortium, and found that >1000 variants (3.6%) within or adjacent to 2393 assayed human exons led to almost complete loss of exon recognition. While most variants at the canonical splice site disrupt splicing, they represent <20% of splice-disrupting variants overall because genetic variation elsewhere dominates. Our results indicate that loss of exon recognition caused by rare genetic variation may play a larger role in trait diversity than previously appreciated, and that MFASS may provide a scalable way to functionally test such variants.