cPRIME enables the systematic mapping of genetic variant effects on RNA stability
Synonymous marker edits significantly improve the detection of rare prime editing events by enhancing signal-to-background ratios. Using two chronic myeloid leukemia cell lines, KBM7 and K562, we performed prime editing screens targeting NF1 exon 27. A pegRNA library, including 98 variants (missense, nonsense and synonymous), was introduced into cells expressing prime editors. Incorporating synonymous marker edits enabled nearly 90% of edits to be detected above background, compared to only 10% without markers. Editing efficiency was site-specific, influenced by poly-T stretches in pegRNAs and SPACER sequences, offering insights for optimizing editing strategies. We further developed cPRIME, a platform that integrates genomic DNA (gDNA) and reverse transcribed RNA (cDNA) readouts to assess the impact of genetic variants on RNA stability. Variants introducing STOP codons reduced cDNA editing frequencies, consistent with nonsense-mediated decay, while a subset of non-STOP variants also showed reduced RNA stability. Three variants, E1206E, R1207R, and D1217E, were prioritized for further analysis based on significant reductions in RNA stability in both cell lines. These findings highlight the utility of synonymous marker edits and cPRIME for precise editing quantification and variant characterization.