cPRIME enables endogenous mapping of genetic variant effects at RNA level.

Understanding the functional consequences of genetic variants, particularly those classified as variants of uncertain significance (VUS), is a major challenge in genomics. While variant effects at protein level are well recognized, roles in post-transcriptional regulation are increasingly appreciated. Here, we introduce cPRIME (prime editing with cDNA readout), a scalable method for assessing variant effects at RNA level. cPRIME employs prime editing to insert genetic variants into endogenous genomic loci, followed by the quantification of changes in variant frequencies at RNA (cDNA) and genomic DNA (gDNA) level. We demonstrate its effectiveness across diverse karyotypes and genomic loci and reveal that synonymous marker edits - frequently used in prime editing screens - can themselves alter RNA processing and thereby confound the interpretation of variant effects. Taken together, cPRIME provides a platform for studying the post-transcriptional consequences of genetic variants in their genomic context and establishes a framework for the functional interpretation of RNA-altering variants in cancer and other diseases.