Development of a A-to-Y and G-to-Y dual base editor and a hypermutator mutating all four bases concurrently

Dual base editors and hyper mutator have been reported for creating or reverting multi-nucleotide variants (MNV) and saturating mutagenesis. Herein, we developed a new type of compact dual base editor by fusing adenine deaminase TadA, Cas9 nickase and an engineered N-methylpurine DNA glycosylase (MPG) to edit both adenine and guanine concurrently, named A&GBE. As the multi-nucleotide diversification property, we further developed a hypermutator (CRISPR-Cas9 combined multi-effectors mediated mutagenesis, CMEM) through nickase/dead Cas9 fusing with an engineered thymine-DNA glycosylase (TDG), TadA and MPG, and recruiting activation-induced cytidine deaminase (AID) by MS2 modified gRNA. The mutator can mutate on all four bases concurrently within about 100bp region around gRNA target. We envision that the dual base editor A&GBE and derived hypermutator CMEM could be used for generating MNVs and directed evolution in future.