Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference.
Nat Commun
; 15(1): 1697, 2024 Feb 24.
Article
en En
| MEDLINE
| ID: mdl-38402281
ABSTRACT
TadA-derived cytosine base editors (TadCBEs) enable programmable Câ¢G-to-Tâ¢A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual Aâ¢T-to-Gâ¢C editing at certain positions and lower editing efficiencies at some sequence contexts and with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual cytosine and adenine base editor, discovering mutations at N46 and Y73 in TadA that prevent Aâ¢T-to-Gâ¢C editing and improve Câ¢G-to-Tâ¢A editing with expanded sequence-context compatibility, respectively. In E. coli, CBE6 variants offer high Câ¢G-to-Tâ¢A editing and no detected Aâ¢T-to-Gâ¢C editing in any sequence context. In human cells, CBE6 variants exhibit broad Cas domain compatibility and retain low off-target editing despite exceeding BE4max and previous TadCBEs in on-target editing efficiency. Finally, we show that the high selectivity of CBE6 variants is well-suited for therapeutically relevant stop codon installation without creating unwanted missense mutations from residual Aâ¢T-to-Gâ¢C editing.
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1
Banco de datos:
MEDLINE
Asunto principal:
Bacteriófagos
/
Edición Génica
Límite:
Humans
Idioma:
En
Año:
2024
Tipo del documento:
Article