Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.
ABE8e
Base editor
DNA binding
Electrostatic interactions
Genome editing
Thermal stability
Journal
Cellular and molecular life sciences : CMLS
ISSN: 1420-9071
Titre abrégé: Cell Mol Life Sci
Pays: Switzerland
ID NLM: 9705402
Informations de publication
Date de publication:
14 Jun 2024
14 Jun 2024
Historique:
received:
01
03
2024
accepted:
02
05
2024
revised:
28
04
2024
medline:
14
6
2024
pubmed:
14
6
2024
entrez:
14
6
2024
Statut:
epublish
Résumé
Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. 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 (T
Identifiants
pubmed: 38874784
doi: 10.1007/s00018-024-05263-7
pii: 10.1007/s00018-024-05263-7
doi:
Substances chimiques
DNA
9007-49-2
Adenine
JAC85A2161
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
257Subventions
Organisme : National Natural Science Foundation of China
ID : 31971377
Informations de copyright
© 2024. The Author(s).
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