Fig. 4. Diversifying 5′ UTR and promoter by BETTER for regulating gene expression.

a Design of base editing targets by replacing the original 5′ UTR between RBS and start codon and −35 box of the promoter to six consecutive Cs, respectively. The target Cs are underlined. The N20 sequences of gRNA are denoted with the dotted box. PAMs for 5′ UTR and promoter editing are shaded in blue and purple, respectively. The mutated G for the generation of a GGG PAM is highlighted in yellow. The original sequence for gfp expression cassette is shown in Supplementary Fig. 1. b, c Analyses of the 5′ UTR (b) and −35 box (c) libraries by NGS and flow cytometry. Three colonies were used for base editing and the cells were mixed with an equal proportion before extraction of genomic DNAs, PCR amplification, and NGS. The original gfp expression cassette was used as a positive control.