High-efficiency small-molecule controlled base editing for in vivo cancer functional genomics

Cancer functional genomics using CRISPR-base editors (BEs) to alter targeted DNA bases within genes of interest holds great promise for molecular characterization and new target discovery. However, traditional BEs, utilizing intact DNA deaminases as mutators, often suffer from limitations given limited control and nonspecific toxicities. These challenges can dampen variant phenotype strength and pose significant barriers to in vivo screening. Here, we developed a small molecule-controllable platform using split-engineered BEs (seBEs) that enables identification of critical residues in cancer targets for in vivo functional genomics screens. By placing deaminase activity under small molecule control, seBE significantly reduced cellular toxicity, and newly enabled robust and controllable functional genomics screens. High-density seBE genetic screens using ~11,000 sgRNAs in vitro and ~ 3700 sgRNAs in vivo, reveal known and previously unknown loss-of-function and dominant negative mutations in cancer therapeutic targets. A deeper tiling seBE screen against ADAR1, a key mediator in cancer immunotherapy, reveals critical residues within functional domains that show no phenotype in vitro, but distinctively elicit non-cell-autonomous cancer dependencies in vivo. Overall, our seBE platform offers a generalizable, controllable, and highly efficient method to systematically identify key residues in cancer functional genomics. Overall design: To further assess possible mechanisms for BE-related toxicity related to effects on gene expression, RNA-seq analysis was performed on cells lentivirally transduced with either nCas9, intact BE with catalytic mutation of the DNA deaminase, intact BE with or without Rap, or seBE with or without Rap.