Mutation-induced penicillin binding protein 2A inactivation through CRISPR-dCas9 gene editing in methicillin-resistant S. aureus
Abstract
Background and Objectives: The global rise of antimicrobial resistance threatens effective treatment of bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen whose resistance to β-lactam antibiotics is primarily mediated by the mecA gene, encoding the low-affinity penicillin-binding protein PBP2a. This study aimed to investigate whether CRISPR-dCas9-mediated targeting of mecA could suppress resistance-associated gene expression and restore β-lactam susceptibility in MRSA.
Materials and Methods: Seven target sites within mecA, comprising two non-coding and five coding regions, were selected for CRISPR-dCas9 targeting. The effects of gene interference were evaluated using antibiotic disk diffusion assays, minimum inhibitory concentration (MIC) testing with gradient E-strips, RT-PCR, and Sanger sequencing.
Results: Targeting non-coding regions suppressed mecA transcription and reduced PBP2a production, whereas targeting coding regions generated mutations associated with dysfunctional PBP2a. CRISPR-dCas9 treatment significantly reduced mecA expression and increased MRSA susceptibility to β-lactam antibiotics, particularly ampicillin and cefixime. MIC values decreased by up to 12-fold and 6-fold, respectively (p<0.05).
Conclusion: CRISPR-dCas9-mediated targeting of mecA effectively reduced β-lactam resistance in MRSA. This approach demonstrates potential for precision-based antimicrobial resistance management and warrants further investigation as a strategy for combating drug-resistant bacterial infections.
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11. Cai R, Lv R, Shi X, Yang G, Jin J. CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation. Int J Mol Sci 2023; 24: 14865.
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13. Tao S, Chen H, Li N, Liang W. The Application of the CRISPR-Cas System in Antibiotic Resistance. Infect Drug Resist 2022; 15: 4155-4168.
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15. Ates A, Tastan C, Ermertcan S. CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus. CRISPR J 2024; 7: 374-384.
16. Hussen BM, Najmadden ZB, Abdullah SR, Rasul MF, Mustafa SA, Ghafouri-Fard S, et al. CRISPR/Cas9 gene editing: a novel strategy for fighting drug resistance in respiratory disorders. Cell Commun Signal 2024; 22: 329.
17. Wessels HH, Méndez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana NE. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat Biotechnol 2020; 38: 722-727.
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21. Yang Y, Liu M, Wang T, Wang Q, Liu H, Xun L, et al. An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency. Microbiol Spectr 2022; 10(6): e0249722.
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24. Hasan M, Ahn J. Evolutionary Dynamics between Phages and Bacteria as a Possible Approach for Designing Effective Phage Therapies against Antibiotic-Resistant Bacteria. Antibiotics (Basel) 2022; 11: 915.
25. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 2012; 12: 177-186.
26. Zhang YM, Zheng L, Xie K. CRISPR/dCas9-Mediated Gene Silencing in Two Plant Fungal Pathogens. mSphere 2023; 8(1): e0059422.
27. Wang J, Liu F, Long J, Jin Y, Chen S, Duan G, Yang H. The application of CRISPR-Cas system in Staphylococcus aureus infection. Heliyon 2024; 10(14): e34383.
28. Garoy EY, Gebreab YB, Achila OO, Tekeste DG, Kesete R, Ghirmay R, et al. Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence and Antimicrobial Sensitivity Pattern among Patients-A Multicenter Study in Asmara, Eritrea. Can J Infect Dis Med Microbiol 2019; 2019: 8321834.
29. Liu Q, Zhuo R, He W, Li C. The new SCCmec type methicillin-resistant Staphylococcus aureus carried CRISPR-cas system isolated from a pig in China. Microb Pathog 2025; 207: 107943.
30. Mougiakos I, Bosma EF, de Vos WM, van Kranenburg R, van der Oost J. Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit. Trends Biotechnol 2016; 34: 575-587.
31. Park JY, Moon BY, Park JW, Thornton JA, Park YH, Seo KS. Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus. Sci Rep 2017; 7: 44929.
32. Basit A, Zhu J, Zheng W. Assessing off-target effects in CRISPR/Cas9: challenges and strategies for precision DNA editing. Arch Microbiol 2026; 208: 114.
33. Wang Z, He Z, Liu Z, Qu M, Gao C, Wang C, et al. A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system. Plant Physiol 2023; 191: 1505-1519.
34. Mazumder I, Rehman M, Deshmukh F, Shah S, Singh A, Ramaiah S, et al. A review on novel strategies to combat multidrug resistance in pathogenic bacteria exploiting synergism between essential oil and antibiotics. World J Microbiol Biotechnol 2026; 42: 36.
2. World Health Organization (2025). Global antibiotic resistance surveillance report 2025: WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS). World Health Organization. Geneva, Switzerland [cited 2026 Sep 2]. Available from: https://iris.who.int/handle/10665/383106
3. Kariuki S. Global burden of antimicrobial resistance and forecasts to 2050. Lancet 2024; 404: 1172-1173.
4. Lade H, Kim JS. Molecular Determinants of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus (MRSA): An Updated Review. Antibiotics (Basel) 2023; 12: 1362.
5. Hidron AI, Low CE, Honig EG, Blumberg HM. Emergence of community-acquired meticillin-resistant Staphylococcus aureus strain USA300 as a cause of necrotising community-onset pneumonia. Lancet Infect Dis 2009; 9: 384-392.
6. Idrees MM, Saeed K, Shahid MA, Akhtar M, Qammar K, Hassan J, et al. Prevalence of mecA- and mecC-Associated Methicillin-Resistant Staphylococcus aureus in Clinical Specimens, Punjab, Pakistan. Biomedicines 2023; 11: 878.
7. Dhungel S, Rijal KR, Yadav B, Dhungel B, Adhikari N, Shrestha UT, et al. Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence, Antimicrobial Susceptibility Pattern, and Detection of mecA Gene among Cardiac Patients from a Tertiary Care Heart Center in Kathmandu, Nepal. Infect Dis (Auckl) 2021; 14: 11786337211037355.
8. Murugaiyan J, Kumar PA, Rao GS, Iskandar K, Hawser S, Hays JP, et al. Progress in Alternative Strategies to Combat Antimicrobial Resistance: Focus on Antibiotics. Antibiotics (Basel) 2022; 11: 200.
9. Darby EM, Trampari E, Siasat P, Gaya MS, Alav L, Webber MA, et al. Molecular mechanisms of antibiotic resistance revisited. Nat Rev Microbiol 2023;21, 280-295.
10. Chow RD, Chen JS, Shen J, Chen S. A web tool for the design of prime-editing guide RNAs. Nat Biomed Eng 2021; 5: 190-194.
11. Cai R, Lv R, Shi X, Yang G, Jin J. CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation. Int J Mol Sci 2023; 24: 14865.
12. Villiger L, Joung J, Koblan L, Weissman J, Abudayyeh OO, Gootenberg JS. CRISPR technologies for genome, epigenome and transcriptome editing. Nat Rev Mol Cell Biol 2024; 25: 464-487.
13. Tao S, Chen H, Li N, Liang W. The Application of the CRISPR-Cas System in Antibiotic Resistance. Infect Drug Resist 2022; 15: 4155-4168.
14. Fri J, Njom HA, Ateba CN, Ndip RN. Antibiotic Resistance and Virulence Gene Characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Healthy Edible Marine Fish. Int J Microbiol 2020; 2020: 9803903.
15. Ates A, Tastan C, Ermertcan S. CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus. CRISPR J 2024; 7: 374-384.
16. Hussen BM, Najmadden ZB, Abdullah SR, Rasul MF, Mustafa SA, Ghafouri-Fard S, et al. CRISPR/Cas9 gene editing: a novel strategy for fighting drug resistance in respiratory disorders. Cell Commun Signal 2024; 22: 329.
17. Wessels HH, Méndez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana NE. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat Biotechnol 2020; 38: 722-727.
18. Chuai G, Ma H, Yan J, Chen M, Hong N, Xue D, et al. DeepCRISPR: optimized CRISPR guide RNA design by deep learning. Genome Biol 2018; 19: 80.
19. Avilan L. Assembling Multiple Fragments: The Gibson Assembly. Methods Mol Biol 2023; 2633: 45-53.
20. Dubnau D, Blokesch M. Mechanisms of DNA Uptake by Naturally Competent Bacteria. Annu Rev Genet 2019; 53: 217-237.
21. Yang Y, Liu M, Wang T, Wang Q, Liu H, Xun L, et al. An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency. Microbiol Spectr 2022; 10(6): e0249722.
22. Bogožalec Košir A, Lužnik D, Tomič V, Milavec M. Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Biosensors (Basel) 2023; 13: 463.
23. Fergestad ME, Stamsås GA, Morales Angeles D, Salehian Z, Wasteson Y, Kjos M. Penicillin-binding protein PBP2a provides variable levels of protection toward different β-lactams in Staphylococcus aureus RN4220. Microbiologyopen 2020; 9(8): e1057.
24. Hasan M, Ahn J. Evolutionary Dynamics between Phages and Bacteria as a Possible Approach for Designing Effective Phage Therapies against Antibiotic-Resistant Bacteria. Antibiotics (Basel) 2022; 11: 915.
25. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 2012; 12: 177-186.
26. Zhang YM, Zheng L, Xie K. CRISPR/dCas9-Mediated Gene Silencing in Two Plant Fungal Pathogens. mSphere 2023; 8(1): e0059422.
27. Wang J, Liu F, Long J, Jin Y, Chen S, Duan G, Yang H. The application of CRISPR-Cas system in Staphylococcus aureus infection. Heliyon 2024; 10(14): e34383.
28. Garoy EY, Gebreab YB, Achila OO, Tekeste DG, Kesete R, Ghirmay R, et al. Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence and Antimicrobial Sensitivity Pattern among Patients-A Multicenter Study in Asmara, Eritrea. Can J Infect Dis Med Microbiol 2019; 2019: 8321834.
29. Liu Q, Zhuo R, He W, Li C. The new SCCmec type methicillin-resistant Staphylococcus aureus carried CRISPR-cas system isolated from a pig in China. Microb Pathog 2025; 207: 107943.
30. Mougiakos I, Bosma EF, de Vos WM, van Kranenburg R, van der Oost J. Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit. Trends Biotechnol 2016; 34: 575-587.
31. Park JY, Moon BY, Park JW, Thornton JA, Park YH, Seo KS. Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus. Sci Rep 2017; 7: 44929.
32. Basit A, Zhu J, Zheng W. Assessing off-target effects in CRISPR/Cas9: challenges and strategies for precision DNA editing. Arch Microbiol 2026; 208: 114.
33. Wang Z, He Z, Liu Z, Qu M, Gao C, Wang C, et al. A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system. Plant Physiol 2023; 191: 1505-1519.
34. Mazumder I, Rehman M, Deshmukh F, Shah S, Singh A, Ramaiah S, et al. A review on novel strategies to combat multidrug resistance in pathogenic bacteria exploiting synergism between essential oil and antibiotics. World J Microbiol Biotechnol 2026; 42: 36.
| Files | ||
| Issue | Vol 18 No 5 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v18i5.22865 | |
| Keywords | ||
| Methicillin resistant Staphylococcus aureus MRSA Penicillin-binding proteins CRISPR-Cas systems Gene editing Drug resistance Microbial antibiotic resistance | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Khan A, Iqbal A, Athar H, Sheikh A. Mutation-induced penicillin binding protein 2A inactivation through CRISPR-dCas9 gene editing in methicillin-resistant S. aureus. Iran J Microbiol. 2026;18(5):741-751.
