Fig. 3

nCBE mediated multiplex and iterative editing. a The diagrammatic sketch of multiple sgRNA assembly. White, grey, and black colored box represented the overlap sequence between every two sgRNAs. b The pie charts of the screening results for editing triple and quadruple target. Blank, orange, green, and blue colored sector represented the none, single, double, and triple target mutants, respectively. The number in the parentheses meant the matched colonies in each group. c The flow chart of plasmid curing. Rectangle, square, and circle represented strain, chromosome, and plasmid, respectively. The yellow and pink colored box on the plasmid represented the sgRNA cassette. Star mark indicated the potential deamination sites. d The colony PCR (25 cycles) using the primers binding to the plasmid (Kana and Cas9) or chromosome (rpoZ). e The pigment phenotype of the WT, single target mutants (∆appA1, ∆ppsR1, and ∆crtB1), and double target mutants (∆appA1-∆ppsR1, ∆crtB1-∆appA1, and ∆crtB1-∆ppsR1) basing on the genetic background of streaked single target mutant (the plasmid cured ∆appA1 or ∆crtB1 strains)