TABLE 2

Mutation efficiency of the CRISPR-Cas two-plasmid systema

Expt no.Host cellPlasmid pTargetTargeting genome locus of sgRNADonor DNA supplied in pTarget or in PCR fragment (F)Length (bp) of homologous extensions (upstream, downstream)Mutation efficiency (%)bPlasmid pTarget curing efficiency (%)
1MG1655pTargetT-ΔcadAcadApTargetT-ΔcadA523, 28186 ± 4100
2MG1655pTargetT-ΔmaeAΔmaeBmaeA, maeBpTargetT-ΔmaeAΔmaeB250, 55097 ± 4ND
3MG1655pTargetT-ΔcadAΔmaeAΔmaeBcadA, maeA, maeBpTargetT-ΔcadAΔmaeAΔmaeB250, 55047 ± 8ND
4MG1655pTargetT-ΔyjcS::ybasyjcSpTargetT-ΔyjcS::ybaS373, 36092 ± 0ND
5MG1655pTargetT-ΔyjcS:: evgASyjcSpTargetT-ΔyjcS::evgAS373, 36075 ± 18ND
6MG1655pTargetT-ΔmaeB::gltP ΔmaeAmaeB, maeApTargetT-ΔmaeB::gltP ΔmaeA250, 55078 ± 26ND
71655ΔcadAcpTargetT-ΔyjcS::evgASyjcSpTargetT-ΔyjcS::evgAS373, 36092 ± 7100
8MG1655pTargetF-cadAcadAΔcadA (F)523, 28169 ± 4ND
9MG1655pTargetF-yjcSyjcSΔyjcS::evgAS (F)40, 406 ± 4ND
10MG1655pTargetF-yjcSyjcSΔyjcS::evgAS (F)373, 36028 ± 10ND
11MG1655pTargetF-kefB-yjcSkefB, yjcSΔkefB, ΔyjcS::evgAS (F)250, 5500ND
12DSM 13699pTargetT-ΔtkrAtkrApTargetT-ΔtkrA483, 513100 ± 0100
13DSM 13699 ΔtkrAcpTargetT-ΔglkglkpTargetT-Δglk500, 50094 ± 8100
  • a The genome editing was performed with the CRISPR-Cas two-plasmid system with pCAS and pTarget, as shown. ND, not determined.

  • b Determined from triple electroporation experiments by colony PCR from 12 transformants for each mutation (agarose electrophoresis gels of colony PCR and relative sequencing results are shown in Fig. S1 and S2 in the supplemental material).

  • c Second round of genome editing.