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 • Instructions for reporter construct design and splice isoform analysis • Steps for site-directed mutagenesis, reporter assays, and transcript quantification • Guidance on prioritizing candidate splice variants using SpliceAI • Procedures for plasmid DNA delivery into mammalian cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Lukes et al. (Sun,) studied this question.