A dual A-to-Y and G-to-Y base editor and a four-base concurrent hypermutator in mammalian cells.

Current dual base editors and hypermutators exhibit limited types of base conversion and constrained mutational diversity. To address this challenge, we engineer a compact dual base editor "A&GBE" by fusing a deaminase and glycosylase with nickase Cas9 (nCas9) to enable concurrent adenine and cytosine editing. Furthermore, we develop quadruple base mutating modules (QBMM) by fusing nickase or dead Cas9 with engineered thymine-DNA glycosylase (TDG), N-methylpurine DNA glycosylase (MPG) and TadA8e, together with MS2-MCP system recruited activation-induced cytidine deaminase (AID). This QBMM enables simultaneous mutation of all four types of nucleotide bases (A, T, G, C) within an approximately 100 base pair (bp) sequence surrounding the gRNA target site. This platform generates hyper diverse multi-nucleotide variants, establishing a powerful tool for accelerated protein evolution, functional genomics, and disease modeling.