Construction and application of a CRISPR/Cas9-assisted genomic editing system for Corynebacterium glutamicum

AMB Express

Table 4 The results of gene editing

Strains and genes	Mutation length (bp)^a	Results (C/I/T)^b	Efficiency (%)
Gene deletion C. glutamicum ATCC 13032
ldh	568	15/5/20	75.0
eutD	877	19/3/22	86.4
gabP	930	19/4/23	82.6
glnA1	730	13/10/23	56.5
glnA2	728	16/7/23	69.6
alaT	614	17/6/23	73.9
argR	317	6/4/10	60.0
gabTD	1949	6/17/23	26.1
aceAB	3450	10/13/23	43.5
poxB	980	15/8/23	65.2
C. glutamicum ATCC 14067
gabP	930	8/15/23	34.8
C. glutamicum ssp. lactofermentum
gabP	930	2/6/8	25.0
Gene insertion (deletion/insertion)
Φ(PtacM-gdhA1)	166/150	22/1/23	95.7
eutD::speC	877/2261	8/15/23	34.8
gabP::gadB	930/1557	7/16/23	30.4
gabP::gadB2B1^m	930/3167	5/18/23	21.7
eutD::gdhA	877/1459	16/7/23	69.6
alaT::gdhA (pCCG1)	614/1459	6/17/23	26.1
alaT::gdhA (pCCG2)	614/1459	5/18/23	21.7

a, the mutation length is the length of deleted or/and inserted fragments. 166-bp region has been deleted from genome and 150-bp fragment was inserted when replace native gdhA1 promoter in C. glutamicum ATCC 13032; 877-bp region has been deleted from genome and 1459-bp fragment was inserted when insert gdhA into eutD; 877-bp region has been deleted from genome and 2261-bp fragment was inserted when insert speC into eutD; 930-bp region has been deleted from genome and 1557-bp fragment was inserted when insert gadB into gabP; 930-bp region has been deleted from genome and 3167-bp fragment was inserted when insert gene cluster gadB2B1^m into gabP; 614-bp region has been deleted from genome and 1459-bp fragment was inserted when insert gdhA into alaT. The gene speC, gdhA and gadB are all under the control of PtacM promoter; b, T: the total number of colonies for PCR verification, C: the number of correct colonies, I: the number of incorrect colonies. The efficiency was calculated by (C/T) * 100%