Regulated Transformation System (RTS): Sddi-Mediated Programmable Shut-Off and Mode Switching of Base Editors

Orthogonal and externally controllable base editors are essential for safe, multiplex single nucleotide manipulation in vivo. Here we identify 140 aa mini deaminase inhibitors (Sddis) that bind their cognate single stranded DNA deaminases (Sdds) with high affinity and exquisite specificity, occluding the DNA binding face of their cognate Sdds and abolishing C to T activity. Based on these highly specific inhibitors, we engineer an adenine and cytosine base editing Regulated Transformation System (ACBE RTS) in which two inactive Sdds are fused to nCas9 as docking arms, while doxycycline inducible SviSddi-SflSdd (CBE) and cumate inducible Air1Sddi-ABE8e (ABE) fusions provide effector modules. Small-molecule regulation switches the platform among four modes-OFF, CBE, ABE and ACBE-achieving up to 43.4 % C-to-T or 42.9 % A-to-G editing at four endogenous human sites. Incorporating a 4,000 member sgRNA library into MARC 145 cells stably expressing ACBE RTS, a single round screen pinpointed four CD163 point mutations that reduced highly pathogenic PRRSV replication by > 2 log10 and abolished viral antigen signals, demonstrating the power of ACBE-RTS for high content functional genomics and antiviral engineering. Compact and bidirectionally switchable on a single Cas9 scaffold, ACBE RTS establishes a versatile framework for precision therapeutics and multidimensional genetic interrogation, and its modular Sddi-Sdd interface should readily extend to TBE and GBE.