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Plant Science Today (PST)

Review Articles

Vol. 12 No. 2 (2025)

Major CRISPR technologies for plant genome editing – A critical review of the molecular mechanisms

DOI
https://doi.org/10.14719/pst.5312
Submitted
26 September 2024
Published
10-04-2025 — Updated on 23-04-2025
Versions

Abstract

The CRISPR-Cas system, an adaptive immune mechanism in bacteria and archaea, is explored for gene editing in crops. This review critically explores the molecular mechanisms in major CRISPR types commonly used for plant genome editing. CRISPR-Cas9, Cas12a (Cpf1) and type I-D systems are the more recently studied systems frequently employed in plant genome editing. Cas9, a class II effector, has a dual-RNA structure to accurately target and cleave DNA, which makes it highly effective for gene knockouts. Cas12a, in contrast, offers unique features, including the ability to produce staggered DNA cuts and the absence of a requirement for tracrRNA, which broadens its editing capabilities by reducing off-target effects. The lesser-known type I-D system, a multicomplex Cas protein system, shows heritable editing in crops. Each system exhibits specific protospacer adjacent motif (PAM) requirements that influence target specificity. Moreover, base and prime editing expand CRISPR’s potential for precise and multiplex genome editing. This review aims to provide a comprehensive understanding of the mechanisms, potential applications and limitations of these CRISPR-Cas systems to guide researchers in selecting the most suitable tool for precise gene editing in plants. Additionally, delivery methods and ethical considerations are discussed, emphasizing their role in optimizing crop improvement strategies.

References

  1. Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol. 2010;64:475–93. https://doi.org/10.1146/annurev.micro.112408.134123
  2. Horvath P, Barrangou R. CRISPR/Cas, the immune system of Bacteria and Archaea. Science. 2010;327(5962):167–70. https://doi.org/10.1126/science.1179555
  3. Koonin EV, Makarova KS. CRISPR-Cas: An adaptive immunity system in prokaryotes. F1000 Biol Rep. 2009;1:95. https://doi.org/10.3410/B1-95
  4. Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol. 2011;9(6):467–77. https://doi.org/10.1038/nrmicro2577
  5. Liu TY, Doudna JA. Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation. J Biol Chem. 2020;295(42):14473-87. https://doi.org/10.1074/jbc.REV120.007034
  6. Jiang F, Doudna JA. CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys. 2017;46:505–29. https://doi.org/10.1146/annurev-biophys-062215-010822
  7. Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol. 2017;37:67–78. https://doi.org/10.1016/j.mib.2017.05.008
  8. Sashital DG, Wiedenheft B, Doudna JA. Mechanism of foreign DNA selection in a bacterial adaptive immune system. Mol Cell. 2012;46(5):606–15. https://doi.org/10.1016/j.molcel.2012.03.020
  9. Westra ER, Semenova E, Datsenko KA, Jackson RN, Wiedenheft B, Severinov K, et al. Type I-E CRISPR-Cas systems discriminate target from non-target DNA through base pairing-independent PAM Recognition. PLoS Genetics. 2013;9(9):e1003742. https://doi.org/10.1371/journal.pgen.1003742
  10. Westra ER, van Houte S, Gandon S, Whitaker R. The ecology and evolution of microbial CRISPR-Cas adaptive immune systems. Phil Trans R Soc B. 2019;374(1772):20190101. https://doi.org/10.1098/rstb.2019.0101
  11. Hille F, Richter H, Wong SP, Bratovic M, Ressel S, Charpentier E. The biology of CRISPR-Cas: Backward and forward. Cell. 2018;172(6):1239–59. https://doi.org/10.1016/j.cell.2017.11.032
  12. Xu Y, Li Z. CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J. 2020;18:2401–15. https://doi.org/10.1016/j.csbj.2020.08.031
  13. Wada N, Osakabe K, Osakabe Y. Type I-D CRISPR system-mediated genome editing in plants. In: Yang B, Harwood W, Que Q, editors. Plant genome engineering. Methods in Molecular Biology, vol 2653. New York, NY: Humana; 2023. p. 21–38. https://doi.org/10.1007/978-1-0716-3131-7_2
  14. McBride TM, Cameron SC, Fineran PC, Fagerlund RD. The biology and type I/III hybrid nature of type I-D CRISPR–Cas systems. Biochem J. 2023;480:471–88. https://doi.org/10.1042/BCJ20220073
  15. Gostimskaya I. CRISPR–Cas9: A history of its discovery and ethical considerations of its use in genome editing. Biochemistry Moscow. 2022;87:777–88. https://doi.org/10.1134/S0006297922080090
  16. Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 2012;40(12):5569–76. https://doi.org/10.1093/nar/gks216
  17. Liao C, Beisel CL. The tracrRNA in CRISPR biology and technologies. Annu Rev Genet. 2021;55:161-81. https://doi.org/10.1146/annurev-genet-071719-022559
  18. Hillary VE, Ceasar SA. A review on the mechanism and applications of CRISPR/Cas9/Cas12/Cas13/Cas14 proteins utilized for genome engineering. Mol Biotechnol. 2023;65(3):311–25. https://doi.org/10.1007/s12033-022-00567-0
  19. Paul B, Montoya G. CRISPR-Cas12a: Functional overview and applications. Biomed J. 2020;43(1):8–17. https://doi.org/10.1016/j.bj.2019.10.005
  20. Asmamaw M, Zawdie B. Mechanism and applications of CRISPR/Cas-9-mediated genome editing. Biologics. 2021;15:353–61. https://doi.org/10.2147/BTT.S326422
  21. Hillary VE, Ceasar SA. A Review on the mechanism and applications of CRISPR/Cas9/Cas12/Cas13/Cas14 proteins utilized for genome engineering. Mol Biotechnol. 2022;65:311-25. https://doi.org/10.1007/s12033-022-00567-0
  22. Shao M, Xu TR, Chen CS. The big bang of genome editing technology: Development and application of the CRISPR/Cas9 system in disease animal models. Dongwuxue Yanjiu. 2016;37(4):191–204. https://doi.org/10.13918/j.issn.2095-8137.2016.4.191
  23. Nishitani C, Hirai N, Komori S, Wada M, Okada K, Osakabe K, et al. Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific Rep. 2016;6:31481. https://doi.org/10.1038/srep31481
  24. Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 2014;166(3):1292–97. https://doi.org/10.1104/pp.114.247577
  25. McAllister KN, Sorg JA. CRISPR genome editing systems in the genus Clostridium: A timely advancement. J Bacteriol. 2019;201(10):e00219-19. https://doi.org/10.1128/jb.00219-19
  26. Amitai G, Sorek R. CRISPR–Cas adaptation: Insights into the mechanism of action. Nat Rev Microbiol. 2016;14(2):67–76. https://doi.org/10.1038/nrmicro.2015.14
  27. Stella S, Alcón P, Montoya G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature. 2017;546:559–63. https://doi.org/10.1038/nature22398
  28. Lee K, Zhang Y, Kleinstiver BP, Guo JA, Aryee MJ, Miller J, et al. Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol J. 2019;17(2):362–72. https://doi.org/10.1111/pbi.12982
  29. Xu R, Qin R, Li H, Li D, Li L, Wei P, et al. Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol J. 2017;15(6):713–17. https://doi.org/10.1111/pbi.12669
  30. Li G, Zhang Y, Dailey M, Qi Y. Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants. Front Genome Ed. 2023;5:1251903. https://doi.org/10.3389/fgeed.2023.1251903
  31. Endo A, Masafumi M, Kaya H, Toki S. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisellanovicida. Scientific Rep. 2016;6:38169. https://doi.org/10.1038/srep38169
  32. Fonfara I, Richter H, Bratovic M, Le Rhun A, Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 2016;532:517–21. https://doi.org/10.1038/nature17945
  33. Banakar R, Schubert M, Kurgan G, Rai KM, Beaudoin SF, Collingwood MA, et al. Efficiency, specificity and temperature sensitivity of Cas9 and Cas12a RNPs for DNA-free genome editing in plants. Front Genome Ed. 2022;3:760820. https://doi.org/10.3389/fgeed.2021.760820
  34. Liu H, Chen W, Li Y, Sun L, Chai Y, Chen H, et al. CRISPR/Cas9 technology and its utility for crop improvement. Int J Mol Sci. 2022;23(18):10442. https://doi.org/10.3390/ijms231810442
  35. Liu Q, Yang F, Zhang J, Liu H, Rahman S, Islam S, et al. Application of CRISPR/Cas9 in crop quality improvement. Int J Mol Sci. 2021;22(8):4206. https://doi.org/10.3390/ijms22084206
  36. Wu J, Chen C, Xian G, Liu D, Lin L, Yin S, et al. Engineering herbicide-resistant oilseed rape by CRISPR/Cas9-mediated cytosine base-editing. Plant Biotechnol J. 2020;18(9):1857-59. https://doi.org/10.1111/pbi.13368

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