Schematics of base editor-based targeted random mutagenesis (BE-TRM) tools, their design and hypermutation mechanisms. (A) Representative architectures of BE-TRM tools, including cytosine (CBE), adenine (ABE), glycosylase (CGBE/CABE), dual BE (DuBE), and BE-polymerase fusion are shown, along with their major deaminase variants. (B) CBE consists of Cas nickase (for example, nCas9) fused to cytidine deaminase and the uracil glycosylase inhibitor domain (UGI). It binds to a target DNA sequence and hydrolytically deaminates cytosine to uracil (C-to-U), ultimately resulting in a C-to-T substitution. (C) ABE is created by fusing nCas9 with an evolved adenine deaminase (TadA*). The TadA* enzyme deaminates deoxyadenosine to deoxyinosine (A-to-I), guiding DNA repair through the nicked strand, and finally producing A-to-G editing. (D) CGBE consists of nCas9, cytidine deaminase, and DNA repair proteins such as uracil DNA N-glycosylase (UNG) or XRCC, which facilitate abasic site formation at the deaminated C. This leads to C-to-G (in humans) or C-to-A editing (in bacteria). (E) DuBE combines the components of CBE and ABE to perform simultaneous C-to-T and A-to-G editing. (F) The BE-polymerase fusion toolset is designed by fusing cytidine or TadA* variant to T7 RNA polymerase (T7RNAP). It mutates the gene located between the T7 promoter and T7 terminator through multiple rounds, generating targeted hypermutation.
