Generating long deletions across the genome with pooled paired prime editing screens

Abstract Engineered deletions are a powerful probe for studying genome architecture, function, and regulation. Yet, the lack of effective methods to create them in large numbers and at multi-kilobase scale has rendered interrogating all the gigabases of human DNA elusive. Prime editing based approaches are promising contenders to fill this gap, but have not been adopted for large-scale pooled screening so far. Here, we performed the first high-throughput paired prime editing deletion screens. To establish the technology, we generated 3,802 deletions of up to 1.2 Mb in a reporter locus in two human cell lines, and identified key determinants of paired prime editing efficiency to be deletion length, predicted pegRNA efficacy, and contact frequency. We used these learnings to generate therapeutically important deletions at rates that are expected to give clinical benefits for retinitis pigmentosa and Duchenne muscular dystrophy. Combining several technological advances, we then performed the first pooled paired prime editing screen to delete over 10,471 sequences of median length 1.1 kb that target 149 genes across the genome. Our screens were high quality, recovering 75% of positive controls. We discovered 462 non-coding regions with fitness effects, and validated deletions with impact as well as tolerated ones in regions with functional annotations. Our screens are the largest pooled characterization of precise deletions to date, demonstrating that prime deletion screens can perturb the genome at scale, and highlighting that most non-coding DNA around essential genes is dispensable for cell growth in one condition.