Tiled base editing screens uncover rare second-site mutations that rescue a misfolded disease protein
Therapeutic base editing can directly revert ~30% of disease-causing missense mutations to the wild-type sequence. One strategy to broaden its reach is to install second-site corrector mutations that improve protein stability or function across multiple pathogenic alleles. Here, we use tiled adenine and cytosine base editing screens to identify such corrector mutations in NPC1, a lysosomal cholesterol transporter whose misfolding causes Niemann-Pick type C disease. Tiling mutagenesis of the endogenous NPC1 locus in haploid human cells carrying the prevalent NPC1I1061T disease allele, combined with phenotypic reporters for cholesterol transport and protein abundance, uncovered rare intragenic suppressor mutations distributed across multiple domains. Unexpectedly, we identified a promiscuous second-site variant that can correct many different pathogenic NPC1 mutations. These correctors improve protein stability, promote ER exit and lysosomal trafficking, and restore cholesterol clearance in patient-derived fibroblasts and induced neurons. Our results establish in situ base editing screens for the discovery of second-site corrector mutations that are actionable for therapeutic base editing when the original mutation cannot be corrected.