Targeted Elimination of bla_NDM-5 Gene in Escherichia coli by Conjugative CRISPR-Cas9 System

Figures & data

Table 1 Bacterial Strains and Plasmids Used in This Study

Bacterial Strains or Plasmids	Relevant Characteristics	Source or Reference
Bacterial Strains
E. coli DH5α	F-,φ80dlacZΔM15,Δ(lacZYA-argF)U169,deoR, recA1, endA1, hsdR17(rk-, mk+), phoA, supE44,λ-, thi-1, gyrA96, relA1	Laboratory stock
E.coli S17-1	with an integrated RP4-2-Tc::Mu-Km::Tn7	Laboratory stock
E. coli ATCC 25922	Quality control strains for antimicrobial susceptibility	Laboratory stock
E. coli J53	F-ara Δ(lac-proAB) rpsL(Str){φ8θ dlacΔ(lacZ)M15	Laboratory stock
E. coli 01	Isolated with plasmid pNDM-5	Laboratory stock
E. coli 02	J53+pNDM-5 with lncFII-type	In this study
E. coli 03	J53+pUC19-NDM-5	Laboratory stock
E. coli 04	J53+pNDM-5 with lncX3-type	Laboratory stock
Plasmids
pCas9(#42876)	pSC101 ori, Cas9 expression plasmid, Cm^R	Laboratory stock
pBBR1MCS-2	Broad-host-range mobilizable plasmid, oriTRP4, Kan^R	Laboratory stock
pCas9-N	pCas9 with sgRNA targeting blaNDM-5, Cm^R	In this study
pCas9-oriT-N	pCas9 with oriTRP4 and sgRNA targeting blaNDM-5, Cm^R	In this study
pBAD-Cas9-oriT-N	pCas9 with oriTRP4 and sgRNA targeting blaNDM-5, arabinose pBAD Promoter, Cm^R	Laboratory stock

Abbreviations: Cm^R, chloramphenicol-resistant; Kan^R, kanamycin-resistant.

Table 2 Primers Used in This Study

Primer Name	Primer Sequence (5′-3′)
N-F	AAACGATCCAGTTGAGGATCTGGGG
N-R	AAAACCCCAGATCCTCAACTGGATC
DR-F	CACGCATTGATTTGAGTCAG
DR-R	GGTGATGTCGGCGATATAGG
oriT-XbaI-F	CGGTCTAGACAGCTGGCAATTCCGGTTCG
oriT-XbaI-R	CGGTCTAGATGGGTATGTGGTCGAAGGCTG
NDM-5-F	GCTCTAGAATGGCTCCAGATGACAAACAT
NDM-5-R	GCTCTAGATGGGTCGAGGTCAGGATAGG
16S-F	CGGTGAATACGTTCYCGG
16S-R	GGWTACCTTGTTACGACTT
qPCR-NDM-F	TTTGGCGATCTGGTTTTCCG
qPCR-NDM-R	ATCAAACCGTTGGAAGCGAC
pCas9-F	AACACGCATTGATTTGAG
pCas9- R	ATAGGAAGGTATCCGACT

Note: The underline indicates the enzyme cutting site.

Figure 1 Plasmid map of pCas9-oriT and pBAD-Cas9-oriT. (A) Plasmid pCas9-oriT containing Cas9, tracrRNA, crRNA, specific guide RNAs(sgRNA), origin of transfer (oriT_RP4) and chloramphenicol resistance gene (CmR). DR: direct repeats (B) The pBAD-Cas9-oriT containing the Cas9, tracrRNA, crRNA, sgRNA, oriT_RP4, the arabinose inducing promoter pBAD and CmR.

Figure 2 Effect of the pCas9 transformation into E coli harboring different pNDM-5. E. coli 02 bears the plasmid pNDM-5 with IncFII-type. E. coli 03 contains the high copy numbers plasmid pUC19- bla_NDM-5. E. coli 04 bears the plasmid pNDM-5 with IncX3-type. Data points represent the mean value of three biological replicates, in which error bars showing the standard deviation.

Figure 3 Relative copy number of plasmid pNDM-5 at each time point. pCas9-N transformed into competent E. coli 02 served as the experimental groups. Plasmid pCas9 transformed into competent E. coli 02 served as the control group. Data points represent the mean value of three biological replicates, in which the error bars showing the standard deviation.

Figure 4 Confirmation of Cas9 and bla_NDM-5 gene presence in E. coli 02 by PCR amplification with primer pCas9-F/R and NDM-5-F/R. M indicates the 2000 bp marker. (A) Cas9 was detected in the pCas9-oriT-N; (B) bla_NDM-5 was detected in the pCas9-oriT-N.

Figure 5 bla_NDM-5 elimination efficiency with different arabinose concentrations and incubation time. (A) Elimination efficiency of bla_NDM-5 with four different concentrations of arabinose (0.01%, 0.10%, 1.00% and 2.00%) at 6 h of incubation. (B) Elimination efficiencies at induction times of 0, 2, 4, 6, 8 and 16 h with 1.00% arabinose induction. Each experiment was performed in triplicate. Data points represent the mean value of three biological replicates, in which the error bars indicate the standard deviation. *p<0.05, **p<0.01.

Figure 6 E. coli J53 harboring pCas9-N precluded the conjunctive plasmid pNDM-5. E. coli 02 as donor and E. coli J53+pCas9-N or E. coli J53+pCas9 as the recipient strain. Data points represent the mean value of three biological replicates, in which the error bars showing indicate the standard deviation. ****p<0.0001.