Fig. 1: Phage-assisted evolution of DddA-derived cytosine base editor for improved activity and expanded targeting scope.
a, Selection to evolve DdCBE using PANCE and PACE. An AP (purple) contains gIII driven by the T7 promoter. The CP (orange) expresses a T7 RNAP–degron fusion. The evolving T7-DdCBE containing DddA split at G1397 is encoded in the SP (blue). Where relevant, the promoters are indicated. b, A 2-amino-acid linker connects T7 RNAP to the degron. The linker sequence contains cytidines C6 and C7 that are targets for DdCBE editing. The nucleotide at position 8 can be varied to T, A, C or G to form plasmids CP-TCC, CP-ACC, CP-CCC and CP-GCC, respectively. In the absence of target C-to-T editing, expression of degron (brown) results in proteolysis of T7 RNAP (orange) and inhibition of gIII expression. Active T7-DdCBE edits one or both target cytidines to install a stop codon (*) within the linker, thus restoring active T7 RNAP to mediate gIII expression. c, Architecture of T7-DdCBE and the 15-bp target spacing region. Nucleotides corresponding to DNA sequences within T7 RNAP, linker and degron genes are colored in orange, gray and brown, respectively.
