Engineering Compact Base Editors by AlphaFold-Guided Mutation Scan and Escherichia coli-Based Tri-Selection.

Area of Science:

• Molecular Biology
• Biotechnology
• Genomic Engineering

Background:

• APOBEC1-based cytosine base editors are effective but can be limited by size and cytotoxicity.
• The bacterial DNA deaminase toxin SsdAtox offers a smaller alternative but suffers from low efficiency and high toxicity.
• Miniaturization of base editors is crucial for efficient delivery methods.

Purpose of the Study:

• To engineer SsdAtox for improved C-to-T base editing efficiency and reduced cytotoxicity.
• To develop a high-throughput screening platform for optimizing DNA deaminases.
• To create compact and efficient base editors for genomic applications.

Main Methods:

• Structure-guided alanine scanning using AlphaFold and CASTpFold identified key residues for SsdAtox modification.
• Site-saturation mutagenesis and directed evolution using a novel E. coli-based Trinity-Screen platform were employed.
• Combinatorial mutagenesis was used to generate multi-site SsdAtox variants.

Main Results:

• Engineering SsdAtox at the K31 gatekeeping residue significantly increased activity but also indels.
• The Trinity-Screen platform successfully selected for variants with high activity and low indels.
• Optimized SsdAtox variants demonstrated up to 31-fold improvement, outperformed BE4max, and showed lower cytotoxicity.
• The Base Editor Performance Index (BEPI) was defined to standardize performance evaluation.

Conclusions:

• Engineered SsdAtox variants represent a significant advancement in compact base editor technology.
• The Trinity-Screen platform is effective for directed evolution of DNA deaminases.
• Optimized SsdAtox variants offer a promising alternative to larger base editors with improved safety and efficacy profiles.