Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.
Cell Mol Life Sci
; 81(1): 257, 2024 Jun 14.
Article
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| MEDLINE
| ID: mdl-38874784
ABSTRACT
Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the AT base pair to GC. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN
/
Evolución Molecular Dirigida
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Simulación de Dinámica Molecular
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Edición Génica
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Mutación
Idioma:
En
Revista:
Cell Mol Life Sci
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2024
Tipo del documento:
Article
País de afiliación:
China