Massively parallel assessment of human variants with base editor screens

Hanna, Ruth E; Hegde, Mudra; Fagre, Christian R; DeWeirdt, Peter C; Sangree, Annabel K; Szegletes, Zsofia; Griffith, Audrey; Feeley, Marissa N; Sanson, Kendall R; Baidi, Yossef; Koblan, Luke W; Liu, David R; Neal, James T; Doench, John G

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens and identify known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we conduct screens with BH3 mimetics and PARP inhibitors and identify point mutations that confer drug sensitivity or resistance. Finally, we create a library of 52,034 clinically-observed variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.