CRISPR-Based Engineering of Plant-Associated Microbiomes: Challenges and Opportunities

Muhammad Zubair; Muqadas Shahzain; Faiqa Shakeel; Nabi Ullah; Haleema Bibi; Muhammad Zaka Ullah; Abdullah Habib; Syed Muhammad Ahmad Shah

doi:10.70749/ijbr.v3i9.2325

Authors

Muhammad Zubair Department of Botany, University of Science and Technology, Bannu, KP, Pakistan.
Muqadas Shahzain İnstitute of Biological Science, Gomal University, Dera Ismail Khan, KP, Pakistan.
Faiqa Shakeel Faculty of Engineering and Science (FES), University of Greenwich, England, UK.
Nabi Ullah Department of Plant Sciences Quaid-i-Azam University Islamabad, Pakistan.
Haleema Bibi Department of Cereal Crops Research Institute Nowshera, Agriculture Research, KP, Pakistan.
Muhammad Zaka Ullah Department of Botany, University of Botany, University of Agriculture, Faisalabad, Punjab, Pakistan.
Abdullah Habib Department of Molecular Biology, University of Okara, Punjab, Pakistan.
Syed Muhammad Ahmad Shah Department of Biotechnology, COMSATS University Islamabad, Abbottabad Campus, KP, Pakistan.

DOI:

https://doi.org/10.70749/ijbr.v3i9.2325

Keywords:

CRISPR-Cas, Plant-associated Microbiomes, Microbiome Engineering, Agriculture, Sustainability, Gene Editing, Biocontrol, Nutrient Uptake, Stress Tolerance, Biosafety

Abstract

In order to address global issues like food security and climate change, plant-associated microbiomes are essential to crop productivity, resilience, and sustainable agriculture. Microbiome engineering has been transformed by CRISPR-Cas technologies, which allow for precise genetic modifications to improve interactions between microbes and plants. The principles of CRISPR, their uses in modifying the rhizosphere, endosphere, and phyllosphere microbiomes, and their potential to enhance nutrient uptake, stress tolerance, and pathogen control are all thoroughly examined in this review. Along with addressing technical, environmental, and regulatory issues, it also highlights areas where multi-omics, artificial intelligence, and nanotechnology can be integrated. Prospects for the future place a strong emphasis on global cooperation for safe deployment, scalable field applications, and customized microbiomes. This review emphasizes how CRISPR is revolutionizing climate-resilient, sustainable agriculture.

Downloads

Download data is not yet available.

References

1. Calicioglu, O., Flammini, A., Bracco, S., Bellù, L., & Sims, R. (2019). The future challenges of food and agriculture: An integrated analysis of trends and solutions. Sustainability, 11(1), 222.

https://doi.org/10.3390/su11010222

2. Berendsen, R. L., Pieterse, C. M., & Bakker, P. A. (2012). The rhizosphere microbiome and plant health. Trends in Plant Science, 17(8), 478-486.

https://doi.org/10.1016/j.tplants.2012.04.001

3. De Faria, M. R., Costa, L. S., Chiaramonte, J. B., Bettiol, W., & Mendes, R. (2020). The rhizosphere microbiome: Functions, dynamics, and role in plant protection. Tropical Plant Pathology, 46(1), 13-25.

https://doi.org/10.1007/s40858-020-00390-5

4. Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., Van der Oost, J., … Koonin, E. V. (2015). An updated evolutionary classification of CRISPR–cas systems. Nature Reviews Microbiology, 13(11), 722-736.

https://doi.org/10.1038/nrmicro3569

5. Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature Biotechnology, 34(9), 933-941.

https://doi.org/10.1038/nbt.3659

6. Wolt, J. D., Wang, K., & Yang, B. (2015). The regulatory status of genome‐edited crops. Plant Biotechnology Journal, 14(2), 510-518.

https://doi.org/10.1111/pbi.12444

7. Bulgarelli, D., Schlaeppi, K., Spaepen, S., Van Themaat, E. V., & Schulze-Lefert, P. (2013). Structure and functions of the bacterial microbiota of plants. Annual Review of Plant Biology, 64(1), 807-838.

https://doi.org/10.1146/annurev-arplant-050312-120106

8. Tringe, S. G., & Rubin, E. M. (2005). Metagenomics: DNA sequencing of environmental samples. Nature Reviews Genetics, 6(11), 805-814.

https://doi.org/10.1038/nrg1709

9. Jain, S., Jain, J., & Singh, J. (2020). The rhizosphere microbiome: Microbial communities and plant health. Plant Microbiome Paradigm, 175-190.

https://doi.org/10.1007/978-3-030-50395-6_10

10. Zhao, Z., Fernie, A. R., & Zhang, Y. (2025). Engineering nitrogen and carbon fixation for next-generation plants. Current Opinion in Plant Biology, 85, 102699.

https://doi.org/10.1016/j.pbi.2025.102699

11. Hardoim, P. R., Van Overbeek, L. S., & Elsas, J. D. (2008). Properties of bacterial endophytes and their proposed role in plant growth. Trends in Microbiology, 16(10), 463-471.

https://doi.org/10.1016/j.tim.2008.07.008

12. Sessitsch, A., Pfaffenbichler, N., & Mitter, B. (2019). Microbiome applications from lab to Field: Facing complexity. Trends in Plant Science, 24(3), 194-198.

https://doi.org/10.1016/j.tplants.2018.12.004

13. Raaijmakers, J. M., & Mazzola, M. (2016). Soil immune responses. Science, 352(6292), 1392-1393.

https://doi.org/10.1126/science.aaf3252

14. Lawson, C. E., Harcombe, W. R., Hatzenpichler, R., Lindemann, S. R., Löffler, F. E., O’Malley, M. A., García Martín, H., Pfleger, B. F., Raskin, L., Venturelli, O. S., Weissbrodt, D. G., Noguera, D. R., & McMahon, K. D. (2019). Common principles and best practices for engineering microbiomes. Nature Reviews Microbiology, 17(12), 725-741.

https://doi.org/10.1038/s41579-019-0255-9

15. Mus, F., Crook, M. B., Garcia, K., Garcia Costas, A., Geddes, B. A., Kouri, E. D., Paramasivan, P., Ryu, M., Oldroyd, G. E., Poole, P. S., Udvardi, M. K., Voigt, C. A., Ané, J., & Peters, J. W. (2016). Symbiotic nitrogen fixation and the challenges to its extension to Nonlegumes. Applied and Environmental Microbiology, 82(13), 3698-3710.

https://doi.org/10.1128/aem.01055-16

16. Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). Search-and-replace genome editing without double-Strand breaks or donor DNA. Nature, 576(7785), 149-157.

https://doi.org/10.1038/s41586-019-1711-4

17. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-rna–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816-821.

https://doi.org/10.1126/science.1225829

18. Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). Search-and-replace genome editing without double-Strand breaks or donor DNA. Nature, 576(7785), 149-157.

https://doi.org/10.1038/s41586-019-1711-4

19. Abudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P., Han, S., Joung, J., Belanto, J. J., Verdine, V., Cox, D. B., Kellner, M. J., Regev, A., Lander, E. S., Voytas, D. F., Ting, A. Y., & Zhang, F. (2017). RNA targeting with CRISPR–cas13. Nature, 550(7675), 280-284.

https://doi.org/10.1038/nature24049

20. Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., Arkin, A., & Lim, W. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183.

https://doi.org/10.1016/j.cell.2013.02.022

21. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823.

https://doi.org/10.1126/science.1231143

22. Lawson, C. E., Harcombe, W. R., Hatzenpichler, R., Lindemann, S. R., Löffler, F. E., O’Malley, M. A., García Martín, H., Pfleger, B. F., Raskin, L., Venturelli, O. S., Weissbrodt, D. G., Noguera, D. R., & McMahon, K. D. (2019). Common principles and best practices for engineering microbiomes. Nature Reviews Microbiology, 17(12), 725-741.

https://doi.org/10.1038/s41579-019-0255-9

23. Zingaro, K. A., & Papoutsakis, E. T. (2015). Building cellular pathways and programs enabled by the genetic diversity of allo-genomes and meta-genomes. Current Opinion in Biotechnology, 36, 16-31.

https://doi.org/10.1016/j.copbio.2015.08.005

24. Raaijmakers, J. M., & Mazzola, M. (2016). Soil immune responses. Science, 352(6292), 1392-1393.

25. https://doi.org/10.1126/science.aaf3252

26. Nielsen, A. A., Der, B. S., Shin, J., Vaidyanathan, P., Paralanov, V., Strychalski, E. A., Ross, D., Densmore, D., & Voigt, C. A. (2016). Genetic circuit design automation. Science, 352(6281).

https://doi.org/10.1126/science.aac7341

27. Cho, S., Shin, J., & Cho, B. (2018). Applications of CRISPR/Cas system to bacterial metabolic engineering. International Journal of Molecular Sciences, 19(4), 1089.

https://doi.org/10.3390/ijms19041089

28. Sessitsch, A., Pfaffenbichler, N., & Mitter, B. (2019). Microbiome applications from lab to Field: Facing complexity. Trends in Plant Science, 24(3), 194-198.

https://doi.org/10.1016/j.tplants.2018.12.004

29. Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., Verdine, V., Donghia, N., Daringer, N. M., Freije, C. A., Myhrvold, C., Bhattacharyya, R. P., Livny, J., Regev, A., Koonin, E. V., Hung, D. T., Sabeti, P. C., Collins, J. J., & Zhang, F. (2017). Nucleic acid detection with CRISPR-cas13a/C2c2. Science, 356(6336), 438-442.

https://doi.org/10.1126/science.aam9321

30. Del Giovane, S., Bagheri, N., Di Pede, A. C., Chamorro, A., Ranallo, S., Migliorelli, D., Burr, L., Paoletti, S., Altug, H., & Porchetta, A. (2024). Challenges and perspectives of CRISPR-based technology for diagnostic applications. TrAC Trends in Analytical Chemistry, 172, 117594.

https://doi.org/10.1016/j.trac.2024.117594

31. Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh, O. O., Metsky, H. C., Durbin, A. F., Kellner, M. J., Tan, A. L., Paul, L. M., Parham, L. A., Garcia, K. F., Barnes, K. G., Chak, B., Mondini, A., Nogueira, M. L., Isern, S., Michael, S. F., Lorenzana, I., Yozwiak, N. L., … Sabeti, P. C. (2018). Field-deployable viral diagnostics using CRISPR-cas13. Science, 360(6387), 444-448.

https://doi.org/10.1126/science.aas8836

32. Tringe, S. G., & Rubin, E. M. (2005). Metagenomics: DNA sequencing of environmental samples. Nature Reviews Genetics, 6(11), 805-814.

https://doi.org/10.1038/nrg1709

33. Widmer, L. A., & Stelling, J. (2018). Bridging intracellular scales by mechanistic computational models. Current Opinion in Biotechnology, 52, 17-24.

https://doi.org/10.1016/j.copbio.2018.02.005

34. Mus, F., Crook, M. B., Garcia, K., Garcia Costas, A., Geddes, B. A., Kouri, E. D., Paramasivan, P., Ryu, M., Oldroyd, G. E., Poole, P. S., Udvardi, M. K., Voigt, C. A., Ané, J., & Peters, J. W. (2016). Symbiotic nitrogen fixation and the challenges to its extension to Nonlegumes. Applied and Environmental Microbiology, 82(13), 3698-3710.

https://doi.org/10.1128/aem.01055-16

35. Lawson, C. E., Harcombe, W. R., Hatzenpichler, R., Lindemann, S. R., Löffler, F. E., O’Malley, M. A., García Martín, H., Pfleger, B. F., Raskin, L., Venturelli, O. S., Weissbrodt, D. G., Noguera, D. R., & McMahon, K. D. (2019). Common principles and best practices for engineering microbiomes. Nature Reviews Microbiology, 17(12), 725-741.

https://doi.org/10.1038/s41579-019-0255-9

36. Zingaro, K. A., & Papoutsakis, E. T. (2015). Building cellular pathways and programs enabled by the genetic diversity of allo-genomes and meta-genomes. Current Opinion in Biotechnology, 36, 16-31.

https://doi.org/10.1016/j.copbio.2015.08.005

37. Sessitsch, A., Pfaffenbichler, N., & Mitter, B. (2019). Microbiome applications from lab to Field: Facing complexity. Trends in Plant Science, 24(3), 194-198.

https://doi.org/10.1016/j.tplants.2018.12.004

38. Jansson, J. K., & Hofmockel, K. S. (2019). Soil microbiomes and climate change. Nature Reviews Microbiology, 18(1), 35-46.

https://doi.org/10.1038/s41579-019-0265-7

39. Raaijmakers, J. M., & Mazzola, M. (2016). Soil immune responses. Science, 352(6292), 1392-1393.

https://doi.org/10.1126/science.aaf3252

40. Cho, S., Shin, J., & Cho, B. (2018). Applications of CRISPR/Cas system to bacterial metabolic engineering. International Journal of Molecular Sciences, 19(4), 1089.

https://doi.org/10.3390/ijms19041089

41. Lawson, C. E., Harcombe, W. R., Hatzenpichler, R., Lindemann, S. R., Löffler, F. E., O’Malley, M. A., García Martín, H., Pfleger, B. F., Raskin, L., Venturelli, O. S., Weissbrodt, D. G., Noguera, D. R., & McMahon, K. D. (2019). Common principles and best practices for engineering microbiomes. Nature Reviews Microbiology, 17(12), 725-741.

https://doi.org/10.1038/s41579-019-0255-9

42. Arber, W. (2014). Horizontal gene transfer among bacteria and its role in biological evolution. Life, 4(2), 217-224.

https://doi.org/10.3390/life4020217

43. ResearchGate lawsuit, walrus spat and a Second World War shipwreck. (2017). Nature, 550(7675), 162-163.

https://doi.org/10.1038/550162a

44. Widmer, L. A., & Stelling, J. (2018). Bridging intracellular scales by mechanistic computational models. Current Opinion in Biotechnology, 52, 17-24.

https://doi.org/10.1016/j.copbio.2018.02.005

45. Reis, L. A., & Rocha, M. S. (2017). DNA interaction with DAPI fluorescent dye: Force spectroscopy decouples two different binding modes. Biopolymers, 107(5).

https://doi.org/10.1002/bip.23015

46. Widmer, L. A., & Stelling, J. (2018). Bridging intracellular scales by mechanistic computational models. Current Opinion in Biotechnology, 52, 17-24.

https://doi.org/10.1016/j.copbio.2018.02.005

47. ResearchGate lawsuit, walrus spat and a Second World War shipwreck. (2017). Nature, 550(7675), 162-163.

https://doi.org/10.1038/550162a

48. Sessitsch, A., Pfaffenbichler, N., & Mitter, B. (2019). Microbiome applications from lab to Field: Facing complexity. Trends in Plant Science, 24(3), 194-198.

https://doi.org/10.1016/j.tplants.2018.12.004

49. Committee on Gene Drive Research in Non-Human Organisms: Recommendations for Responsible Conduct, Board on Life Sciences, Division on Earth and Life Studies, National Academies of Sciences, Engineering, and Medicine. Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values [Internet]. Washington, D.C.: National Academies Press; 2016 [cited 2025 Sept 24]. Available from:

http://www.nap.edu/catalog/23405

50. Wolt, J. D., Wang, K., & Yang, B. (2015). The regulatory status of genome‐edited crops. Plant Biotechnology Journal, 14(2), 510-518.

https://doi.org/10.1111/pbi.12444

51. Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature Biotechnology, 34(9), 933-941.

https://doi.org/10.1038/nbt.3659