MutaT7GDE: A Single Chimera for the Targeted, Balanced, Efficient, and Processive Installation of All Possible Transition Mutations In Vivo.
ACS Synth Biol
; 13(9): 2693-2701, 2024 Sep 20.
Article
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| MEDLINE
| ID: mdl-39190860
ABSTRACT
Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Virales
/
ARN Polimerasas Dirigidas por ADN
Idioma:
En
Revista:
ACS Synth Biol
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos