Fig. 3

Insertion of the gene upp in a Δupp locus with single point mutation. a Representation of the Δupp locus before (left) and after (right) knock-in of the upp gene. The protospacers recognized by spacers sp4 and sp5 are underlined and highlighted with dark and light brown, respectively, and their PAM is in bold. The 1 kb flanking sites surrounding Δupp are in grey and blue, while the inserted upp sequence is in green; after introduction of the upp sequence, the protospacers sequences are disrupted, and the starting codon ATG is replaced by GTG via a 1 bp substitution (red). b Colony forming units (CFU) obtained after conjugation with pBBR_Cas9 plasmids (pBBR_Cas9_KIupp1000HR_sp4 and pBBR_Cas9_KIupp1000HR) harbouring different Δupp targeting spacers (sp4 and sp5) and 1 kb HR templates for the knock-in of the upp gene accompanied by single nucleotide substitution; the plasmid with the non-targeting spacer (pBBR_Cas9_NT) was used as conjugation control. c Sequence verification of the desired upp insertion and single nucleotide substitution, by sanger sequencing