Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e

Area of Science:

• Molecular Biology
• Biochemistry
• Bioengineering

Background:

• Adenine base editors (ABEs) convert A:T base pairs to G:C using CRISPR Cas nickase and deaminase.
• ABE8e, an evolved variant, exhibits enhanced base editing activity due to eight mutations in its deaminase, TadA8e.
• The functional impact of these specific mutations on ABE8e's performance remains largely uncharacterized.

Purpose of the Study:

• To elucidate the role of directed-evolution mutations in the catalytic activity and stability of adenine base editors.
• To investigate the molecular mechanisms underlying the enhanced DNA-binding affinity and editing efficiency of ABE8e.

Main Methods:

• Molecular dynamics (MD) simulations to analyze DNA-binding interactions and protein dynamics.
• Experimental validation using microscale thermophoresis (MST) to assess DNA-binding affinity.
• In vivo reversion mutation experiments to confirm the functional significance of identified mutations.

Main Results:

• MD simulations revealed that TadA8e exhibits higher DNA-binding affinity than TadA7.10, driven by increased positive charge density in the DNA-binding region.
• Mutations R111, N119, and N167 were identified as critical for enhanced DNA binding, confirmed by MST and in vivo experiments.
• Directed evolution mutations significantly improved the thermal stability of both TadA8e (by ~12°C) and ABE8e (by ~9°C).

Conclusions:

• Directed-evolution mutations enhance ABE8e's substrate-binding ability through improved electrostatic interactions with DNA.
• These mutations also confer increased protein stability, contributing to the overall improved performance of ABE8e.
• The findings provide a rational basis for further optimization of adenine base editing systems.