Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1T via CRISPR/Cas9 with Red System
Abstract
:1. Introduction
2. Results
2.1. Construction and Screening of the CRISPR/Cas9-Based Genome Editing System in the X1T Strain
2.2. Evaluation of the Efficiency of Gene Knockout of opdB in the X1T Strain
2.3. Plasmid Curing
2.4. Assessment of the Degradation Capacity of OPs in the X1T-ΔopdB Strain
3. Discussion
4. Material and Methods
4.1. Strains and Plasmids
4.2. Culture and Reagents
4.3. Construction of the Prokaryotic Expression Vector
4.4. Genome Editing
4.5. Plasmid Curing
4.6. Degradation Experiments
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Name | Sequence (5′-3′) | Purpose | |
---|---|---|---|
Oligos | dsgRNA-F | gtgg ATTGAAGCCGTTCGCCCGGA GGTCGCGGGCAAGTCGTATC | The dual sgRNA in CRISPR vector |
dsgRNA-R | aaac GATACGACTTGCCCGCGACC TCCGGGCGAACGGCTTCAAT | ||
T600-F | tgtccatacccatgg TCTAGA TCCGAGTCCGCCAGGACAATGT | The upstream homology arm for opdB deletion | |
T600-R | actgaagaacttgaac TGTTGTTTCCTTTGAATTTAGGAAAGG | ||
B600-F | ttcaaaggaaacaaca GTTCAAGTTCTTCAGTTCGCTCC | The downstream homology arm for opdB deletion | |
B600-R | gggagtatgaaaagt CTCGAG GTTTTCCCGCAAAGGAAACTCCGA | ||
P1 | AGTAGAAACAGACGAAGAATCCAT | Detection of homology arms and sgRNA | |
P2 | TCTGAATGGCGGGAGTATGAAAAGT | ||
opdB-F | ATGAAGACAAGCGTTCATCTCAGTT | The degradation gene of organophosphorus insecticides | |
opdB-R | TTACTGTTGCAGAGCAGATGCCACA | ||
Neo-F | ATGATTGAACAAGATGGATTGCA | Neomycin resistance gene in CRISPR vector | |
Neo-R | TCAGAAGAACTCGTCAAGAAGGC | ||
Cas9-F | ATGGATAAGAAATACTCAATAGGCTTAG | Expression of Cas9 protein encoded by cas9 gene | |
Cas9-R | TCAGTCACCTCCTAGCTGACTCAAA |
Name | Sequence (5′-3′) | Position in an Original Plasmid pX1 | |
---|---|---|---|
Spacers | sgRNA-188rev | ATTGAAGCCGTTCGCCCGGA GGG | 7488–7466 |
sgRNA-1016fw | GGTCGCGGGCAAGTCGTATC TGG | 8274–8296 |
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OPs | Concentration of OPs after 24 h of Treatment (mg/L) | ||
---|---|---|---|
Control | C. nantongensis X1T | C. nantongensis X1T-ΔopdB | |
Profenofos | 17.1 ± 0.4 c | 9.7 ± 0.2 a | 17.7 ± 0.3 c |
Chlorpyrifos | 19.1 ± 0.1 ab | ND | 19.4 ± 0.4 ab |
Methyl parathion | 19.6 ± 0.0 a | ND | 19.7 ± 0.4 a |
Parathion | 18.7 ± 0.5 b | ND | 18.7 ± 0.1 b |
Triazophos | 18.6 ± 0.2 b | 1.4 ± 0.1 c | 18.6 ± 0.2 b |
Phoxim | 18.5 ± 0.4 b | 1.0 ± 0.0 d | 18.7 ± 0.3 b |
Fenitrothion | 17.6 ± 0.4 c | ND | 17.0 ± 0.4 c |
Isocarbophos | 14.2 ± 0.3 d | 6.2 ± 0.1 b | 14.0 ± 0.2 d |
Strains and Plasmids | Description | Source |
---|---|---|
E. coli DH5α | supE44ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 | Solarbio |
pMD19-T | Apr | Takara |
Cupriavidus nantongensis X1T | an aerobic, Gram-negative, motile Proteobacterium that forms circular colonies | Lab store |
pCas9 | Cmr, bacterial expression of Cas9 nuclease; a temperature sensitive vector in E. coli for genome editing | BioSci |
pKD20 | the Red recombination system and the arabinose-inducible ParaB promoter | BioSci |
pDN19 | Tcr, the P. aeruginosa shuttle cloning vector | BioSci |
pALB2 | Tcr, SacB | BioSci |
pAK1900 | Apr, broad-host-range cloning vector | BioSci |
pAK405 | Plasmid for allelic exchange and seamless gene deletions; Nmr | BioSci |
pACasN | A shuttle vector; Tcr, SacB, Cas9 nuclease and the Red recombination system | This study |
pDCRH | A shuttle vector; Nmr; SacB | This study |
pDCRH-spacer | The pDCRH plasmid with the dual sgRNA | This study |
pDCRH-ΔUD | Digested pDCRH-spacer plasmid | This study |
pDCRH-UD | The pDCRH plasmid with the dual sgRNA and homologous arms | This study |
X1-pACasN | Tcr | This study |
X1-ΔopdB | The opdB gene deleted in Cupriavidus nantongensis X1T | This study |
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Zhang, Y.; Geng, Y.; Li, S.; Shi, T.; Ma, X.; Hua, R.; Fang, L. Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1T via CRISPR/Cas9 with Red System. Int. J. Mol. Sci. 2023, 24, 6003. https://doi.org/10.3390/ijms24066003
Zhang Y, Geng Y, Li S, Shi T, Ma X, Hua R, Fang L. Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1T via CRISPR/Cas9 with Red System. International Journal of Molecular Sciences. 2023; 24(6):6003. https://doi.org/10.3390/ijms24066003
Chicago/Turabian StyleZhang, Yufei, Yuehan Geng, Shengyang Li, Taozhong Shi, Xin Ma, Rimao Hua, and Liancheng Fang. 2023. "Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1T via CRISPR/Cas9 with Red System" International Journal of Molecular Sciences 24, no. 6: 6003. https://doi.org/10.3390/ijms24066003