TABLE 2.

Mutation efficiency of the CRISPR-Cas two-plasmid system^a

Expt no.	Host cell	Plasmid pTarget	Targeting genome locus of sgRNA	Donor DNA supplied in pTarget or in PCR fragment (F)	Length (bp) of homologous extensions (upstream, downstream)	Mutation efficiency (%)b	Plasmid pTarget curing efficiency (%)
1	MG1655	pTargetT-ΔcadA	cadA	pTargetT-ΔcadA	523, 281	86 ± 4	100
2	MG1655	pTargetT-ΔmaeAΔmaeB	maeA, maeB	pTargetT-ΔmaeAΔmaeB	250, 550	97 ± 4	ND
3	MG1655	pTargetT-ΔcadAΔmaeAΔmaeB	cadA, maeA, maeB	pTargetT-ΔcadAΔmaeAΔmaeB	250, 550	47 ± 8	ND
4	MG1655	pTargetT-ΔyjcS::ybas	yjcS	pTargetT-ΔyjcS::ybaS	373, 360	92 ± 0	ND
5	MG1655	pTargetT-ΔyjcS:: evgAS	yjcS	pTargetT-ΔyjcS::evgAS	373, 360	75 ± 18	ND
6	MG1655	pTargetT-ΔmaeB::gltP ΔmaeA	maeB, maeA	pTargetT-ΔmaeB::gltP ΔmaeA	250, 550	78 ± 26	ND
7	1655ΔcadAc	pTargetT-ΔyjcS::evgAS	yjcS	pTargetT-ΔyjcS::evgAS	373, 360	92 ± 7	100
8	MG1655	pTargetF-cadA	cadA	ΔcadA (F)	523, 281	69 ± 4	ND
9	MG1655	pTargetF-yjcS	yjcS	ΔyjcS::evgAS (F)	40, 40	6 ± 4	ND
10	MG1655	pTargetF-yjcS	yjcS	ΔyjcS::evgAS (F)	373, 360	28 ± 10	ND
11	MG1655	pTargetF-kefB-yjcS	kefB, yjcS	ΔkefB, ΔyjcS::evgAS (F)	250, 550	0	ND
12	DSM 13699	pTargetT-ΔtkrA	tkrA	pTargetT-ΔtkrA	483, 513	100 ± 0	100
13	DSM 13699 ΔtkrAc	pTargetT-Δglk	glk	pTargetT-Δglk	500, 500	94 ± 8	100

^aThe genome editing was performed with the CRISPR-Cas two-plasmid system with pCAS and pTarget, as shown. ND, not determined.

^bDetermined 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).

^cSecond round of genome editing.