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

Area of Science:

• Molecular Biology
• Gene Editing
• Biotechnology

Background:

• Orthogonal and controllable base editors are essential for safe in vivo single-nucleotide manipulation.
• Existing base editing technologies require improvement for precise control and multiplexing.

Purpose of the Study:

• To engineer a novel base editing system (ACBE-RTS) with external control for multiplexed single-nucleotide editing.
• To demonstrate the system's efficacy in achieving precise C-to-T and A-to-G base conversions.
• To utilize the system for genetic screening to identify host factors involved in viral replication.

Main Methods:

• Identification of miniature deaminase inhibitors (Sddis) that bind single-stranded DNA deaminases (Sdds).
• Engineering of the ACBE-RTS platform using inactive dSdds fused to nCas9 and inducible effector modules (CBE and ABE).
• Small-molecule inducible switching between four editing modes (OFF, CBE, ABE, ACBE).
• Application of ACBE-RTS in a genetic screen using a large sgRNA library in MARC-145 cells.

Main Results:

• The ACBE-RTS platform achieved up to 43.4% C-to-T and 42.9% A-to-G editing at endogenous human sites.
• Small-molecule regulation allowed seamless switching between editing modes.
• Genetic screening identified four key amino acids in monkey CD163 that significantly reduced PRRSV replication (>100-fold).

Conclusions:

• ACBE-RTS provides a compact, versatile, and switchable framework for precision therapeutics and genetic interrogation.
• The modular Sddi-Sdd interface offers potential for extension to other base editors (TBE, GBE).
• The identified CD163 variants demonstrate a novel strategy for controlling viral replication.