PETRA: Prime editing of transcribed regulatory elements to assay expression

Predicting how changes in human DNA sequence impact gene expression remains challenging. Here, we present PETRA ( P rime E diting of T ranscribed R egulatory elements to A ssay expression), a multiplexed genome editing method to quantify the effects of regulatory variants at scale. PETRA leverages the delivery of variants to abundantly transcribed regions of genes such that sequence-specific effects on mRNA expression can be read out by amplicon sequencing. We demonstrate PETRA in Jurkat cells by scoring 13,935 six-nucleotide insertions delivered to the 5’ untranslated regions (5’ UTRs) of four genes important for T cell responses, namely VAV1 , IL2RA , CD28 and OTUD7B . Effects on expression are linked to the creation of new transcription factor binding sites (TFBSs), as well as to alterations in splicing and translation initiation. Combinatorial delivery of TFBSs identified using PETRA enables the discovery of alleles that increase mRNA expression more than 10-fold. Lastly, we extend PETRA to primary human T cells and compare variant effects across cell types. These experiments establish PETRA as a flexible means of dissecting the logic of gene regulation across genomic contexts and cell types.