Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Abstract

Base editing (BE) can be applied to characterize single nucleotide variants (SNVs) of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular BE-activity ‘sensors’ that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor–sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated SNVs in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models, and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput BE screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.