Regulated transformation system (RTS): sddi-mediated programmable shut-off and mode switching of base editors

Orthogonal and externally controllable base editors are critical for safe multiplexed single-nucleotide manipulation in vivo. Here, we identify ~140-aa miniature deaminase inhibitors (Sddis) that bind cognate single-stranded DNA deaminases (Sdds) with high affinity and specificity, occluding their DNA-binding surfaces to completely inhibit C-to-T activity. Based on these inhibitors, we engineer an adenine and cytosine base editing-regulated transformation system (ACBE-RTS). This platform features two inactive dSdds fused to nCas9 as docking arms, with effector modules provided by doxycycline-inducible SviSddi-SflSdd (CBE) and cumate-inducible Air1Sddi-ABE8e (ABE) fusions. Small-molecule regulation enables switching among four modes (OFF, CBE, ABE, ACBE), achieving up to 43.4% C-to-T or 42.9% A-to-G editing at four endogenous human sites. Using a 4000-member sgRNA library in MARC-145 cells stably expressing ACBE-RTS, a three-round screening identified four key amino acids in monkey CD163 that reduced replication of highly pathogenic PRRSV by >100-fold and eliminated detectable viral-antigen staining. Compact and multi-mode switchable on a single Cas9 scaffold, ACBE-RTS establishes a versatile framework for precision therapeutics and genetic interrogation. Its modular Sddi-Sdd interface could in principle be readily extended to other base editors, such as thymine and guanine base editors (TBE and GBE).