Protocol for validating computationally predicted splice-altering variants using full-length gene reporter assays

Up to half of human disease-causing variants may disrupt RNA splicing, but their computationally predicted effects require experimental validation. Here, we present a protocol to test splice-altering variants using full-length gene reporters. We describe steps for reporter construct design, site-directed mutagenesis, delivery into cells, and isoform analysis. Full-length reporters offer key advantages over minigenes, particularly for clinically relevant genes with compact loci, by preserving native splicing context and enabling more accurate functional interpretation of variants of uncertain significance. For complete details on the use and execution of this protocol, please refer to Kiianitsa et al.¹.