Directed-evolution mutations of adenine base editor ABE8e improve its DNA-binding affinity and protein stability

Adenine base editors (ABEs), consisting of CRISPR-associated (Cas) nickase and deaminase, can convert the A:T base pair to G:C. Previous studies have shown that ABE8e, a directed-evolution variant of ABE7.10 with eight amino-acid mutations in its deaminase TadA-8e, has higher base editing activity than ABE7.10. However, it remains unclear how the directed-evolution mutations of ABE8e increase its editing activity. Here, we combined molecular dynamics (MD) simulations and experimental measurements to elucidate the molecular origin of the activity enhancement by these mutations. MD simulations and microscale thermophoresis measurements showed that TadA-8e has a higher DNA-binding affinity than TadA-7.10, and the main driving force is electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attractions with DNA. We showed that R111 is the key mutation for the enhanced binding to DNA, which explains why the ABE-8e activity was dramatically reduced when R111 was mutated back to the original T. Unexpectedly, we also found that these mutations improve the thermal stability of TadA-8e by ∼12°C (Tm). Our results indicate that the editing activities of ABEs are closely related to their DNA-binding affinity and protein stability, thus providing a rational basis for their optimization. Author Summary Adenine-to-guanine mutations account for 47% of known disease-causing point mutations in humans. Adenine base editors (ABEs), which can restore the guanine mutation to adenine, are a promising tool for precision gene therapy. ABE8e is the most widely used editor today due to its high editing efficacy and was derived from the first-generation base editor ABE7.10 by directed evolution. Compared to ABE7.10, ABE8e contains 8 directed-evolution amino-acid mutations. Understanding how these mutations affect the efficiency of ABE8e is important for the development of efficient base editors. In this study, we combined computational and experimental approaches to investigate how these mutations affect the editing activity of ABE8e. Our results showed that these directed-evolution mutations improve the DNA binding affinity and the protein stability of ABE8e, by enhancing electrostatic and hydrogen bonding interactions. Therefore, our study provides valuable insights for the design of more efficient base editors. ### Competing Interest Statement The authors have declared no competing interest.