High throughput prime editing screens identify functional DNA variants in the human genome

Despite tremendous progress in detecting DNA variants in the human genome, interpreting variants impact remains challenging. We present a new pooled prime-editing screen method, which uses lentiviral libraries and can be applied to characterize genome function for both coding and non-coding sequences. We first identified essential nucleotides for a MYC enhancer by prime editing-enabled saturation mutagenesis. Second, we applied prime editing screens to characterize thousands of breast cancer-associated non-coding variants and uncovered hundreds of functional variants affecting cell fitness. Finally, we demonstrated the utility of prime-editing screens for assessing the functions of clinical variants and revealed mutation consequences for variants of unknown significance. Collectively, we demonstrate that pooled prime editing screens can be used for characterizing genetic variants at the base pair resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic targets.