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Plant Science Today (PST)

Review Articles

Vol. 12 No. Sp2 (2025): Current Trends in Plant Science and Microbiome for Sustainability

CRISPR-Cas9 system: A genome editing tool

DOI
https://doi.org/10.14719/pst.5127
Submitted
18 September 2024
Published
01-07-2025

Abstract

The CRISPR-Cas9 system has become as a groundbreaking tool for modifying genomes, completely changing the field of genetic research and biotechnology. This review article outlines the historical background, constituents and operation of the CRISPR-Cas9 system, emphasizing its exceptional accuracy and adaptability in selectively modifying DNA sequences. Initially discovered as an adaptive immune system in prokaryotes, CRISPR-Cas9 has since been adapted for use in various species, enabling targeted gene editing through gene disruption, insertion and correction. The review examines the technical components of the CRISPR-Cas9 system, including the design of guide RNA, delivery methods and the potential for off-target effects. It also explores recent advancements aimed at enhancing the accuracy and efficiency of this technology. Furthermore, the article discusses the broad applications of CRISPR-Cas9 in fields such as cancer research, gene therapy and agricultural biotechnology, underscoring its potential to provide innovative solutions for genetic disorders and to improve crop resilience. In addition, the review discusses the ethical and regulatory considerations associated with genome editing, emphasizing the significance of responsible and judicious use of this powerful technology. By analysing current research and exploring future directions, this study aims to provide a comprehensive overview of the CRISPR-Cas9 system and its profound impact on science and medicine.

References

  1. 1. Hossain MA. CRISPR-Cas9: A fascinating journey from bacterial immune system to human gene editing. Prog Mol Biol Transl Sci. 2021;178:63–83. https://doi.org/10.1016/bs.pmbts.2021.01.001
  2. 2. Hussen BM. CRISPR/Cas9 gene editing: A novel strategy for fighting drug resistance in respiratory disorders. Cell Commun and Sign. 2024;22(329):1–21. https://doi.org/10.1186/s12964-024-01713-8
  3. 3. Heidenreich M, Zhang F. Applications of CRISPRCas systems in neuroscience. Nat Rev Neurosci. 2016;17:36–44. https://doi.org/10.1038/nrn.2015.2
  4. 4. Lucas D, O’Leary HA, Ebert BL, Cowan CA, Tremblay CS. Utility of CRISPR/Cas9 systems in haematology research. Exp Hematol. 2017;54:1–3. https://doi.org/10.1016/j.exphem.2017.06.006
  5. 5. Biagioni A, Laurenzana A, Margheri F, Chilla A, Fibbi G, Rosso MD. Delivery systems of CRISPR/Cas9- based cancer gene therapy. J Biol Eng. 2018;12(33):1–9. https://doi.org/10.1186/s13036-018-0127-2
  6. 6. Yin H, Xue W, Anderson DG. CRISPR–Cas: A tool for cancer research and therapeutics. Nat Rev Clin Oncol. 2019;16(5):281–95. https://doi.org/10.1038/s41571-019-0166-8
  7. 7. Liu J, Zhou G, Zhang L, Zhao Q. Building potent chimeric antigen receptor T cells with CRISPR genome editing. Front Immunol. 2019:10:456. https://doi.org/10.3389/fimmu.2019.00456
  8. 8. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121),819–23. https://doi.org/10.1126/science.1231143
  9. 9. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNAv endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816–21. https://doi.org/10.1126/science.
  10. 1225829.
  11. 10. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339(6121):823–26. https://doi.org/10.1126/science.1232033
  12. 11. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 2014;343(6176):1247997. https://doi.org/10.1126/science.1247997
  13. 12. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 2014;507(7490):62–67. https://doi.org/10.1038/nature13011
  14. 13. Zuo Z, Liu J. Cas9-catalysed DNA cleavage generates staggered ends: evidence from molecular dynamics simulations. Sci Rep. 2016;6(37584):1–9. https://doi.org/10.1038/srep37584
  15. 14. Lemos BR, Kaplan AC, Bae JE, Ferrazzoli AE, Kuo J, Anand RP, et al. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/ deletion profiles. Proc Natl Acad Sci U S A. 2018;115(9):
  16. E2040–47. https://doi.org/10.1073/pnas.1716855115.
  17. 15. Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014;24(6): 1012–19. https://doi.org/10.1101/gr.171322.113
  18. 16. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 2014;3:e04766. https://doi.org/10.7554/eLife.04766
  19. 17. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015;33(5):538–42. https://doi.org/10.1038/nbt.3190.
  20. 18. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315:1709–12. https://doi.org/10.1126/science.1138140
  21. 19. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015;13(11):722–36. https://doi.org/10.1038/nrmicro3569
  22. 20. Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017;15(3):169–82. https://doi.org/10.1038/nrmicro.2016.184
  23. 21. 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
  24. 22. Oliver AP, Gersbach CA. The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Biol. 2020;20(8):490–507. https://doi.org/10.1038/s41580-019-0131-5
  25. 23. Yoshimi K, Mashimo K. Genome editing technology and applications with the type I CRISPR system. Gen Genom Edit Cell Biol. 2022;3-4:100013. https://doi.org/10.1016/j.ggedit.2022.100013
  26. 24. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014;157:1262–78. https://doi.org/10.1016/j.cell.2014.05.010
  27. 25. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakatura A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isoenzyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987;169:5429–33. https://doi.org/10.1128/jb.169.12.5429-5433.1987
  28. 26. Groenen PM, Bunschoten AE, Soolingen DV, Embden JDV. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol Microbiol. 1993;10(5):1057–65. https://doi.org/10.1111/j.1365-2958.1993.tb00976.x
  29. 27. Hoe N, Nakashima K, Grigsby D, Pan X, Dou SJ, Naidich S, et al. Rapid molecular genetic subtyping of serotype M1 group A Streptococcus strains. Emerg Infect Dis. 1999;5(2):254–63. https://doi.org/10.3201/eid0502.990210
  30. 28. Mojica FJM, Juez G, Rodriguez‐Valera F. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol Microbiol. 1993;9,613–21. https://doi.org/10.1111/j.1365-2958.1993.
  31. tb01721.x
  32. 29. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol. Evol., 2005;60:174–82. https://doi.org/10.1007/s00239-004-
  33. 0046-3
  34. 30. Bolotin A, Quinquis B, Sorokin A, Dusko Ehrlich S. Clustered regularly interspaced short palindrome repeats (CRISPRS) have spacers of extrachromosomal origin. Microbiology 2005;151:2551–61. https://doi.org/10.1099/mic.0.
  35. 28048-0
  36. 31. Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005;151:653–63,. https://doi.org/10.1099/mic.0.27437-0
  37. 32. Popkov V A, Zorova LD, Korvigo IO, Silachev DN, Jankauskas SS, et al. Do mitochondria have an immune system? Biochemistry (Moscow) 2016;81:1229–36. https://doi.org/10.1134/S0006297916100217
  38. 33. Isaev AB, Musharova OS, Severinov KV. Microbial arsenal of antiviral defences. Part I, Biochemistry (Moscow) 2021; 86:319–37.https://doi.org/10.1134/S0006297921030081
  39. 34. Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322:1843–45. https://doi.org/10.1126/science.1165771
  40. 35. Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, et al. Discovery and functional characterization of diverse class 2 CRISPR–Cas systems. Mol Cell 2015;60:385–97. https://doi.org/10.1016/j.molcel.2015.10.008
  41. 36. Shmakov S, Smargon A, Scott D, Cox D, Pyzocha NV, et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat Rev Microbiol 2017;15:169–82. https://doi.org/10.1038/nrmicro.2016.184
  42. 37. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 2016;353:aaf5573. https://doi.org/10.1126/science.aaf5573
  43. 38. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR–Cas13. Nature 2017;550:280–84. https://doi.org/10.1038/nature24049
  44. 39. Liu Z, Dong H, Cui Y, Cong L, Zhang D. Application of different types of CRISPR/Cas-based systems in bacteria. Microbial Cell Factories 2020;19(172):1–14. https://doi.org/10.1186/s12934-020-01431-z
  45. 40. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata S, Dohmae N. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2017;176(12):139–48. https://doi.org/10.1016/j.cell.2014.02.001
  46. 41. Richter C, Gristwood T, Clulow JS, Fineran PC. In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype I-F CRISPR/ Cas system. PLoS ONE 2012;7(12):e49549. https://doi.org/10.1371/journal.pone.0049549
  47. 42. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011;471(7340):602–07. https://doi.org/10.1038/nature09886
  48. 43. Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, et al. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell 2013;50(4):488–503. https://doi.org/10.1016/j.molcel.2013.05.001
  49. 44. Wiedenheft B, Dujin EV, Bultema JB, Waghmare SP, Zhou K, Barendregt A, et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci USA 2011;108(25):10092–97. https://doi.org/10.1073/pnas.1102716108
  50. 45. Brouns SJ, Jore MM, Lundgren M, Westra, ER, Slijkhuis RJ, Snijders A P, et al. Small CRISPR RNAs guide antiviral defence in prokaryotes. Sci 2008:321,960–64. https://doi.org/10.1126/science.1159689
  51. 46. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, et al. Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol 2017;35(8):789–92. https://doi.org/10.1038/nbt.3900
  52. 47. Jore MM, Lundgren M, Duijn EV, Bultema JB, Westra ER, Waghmare SP, et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 2011;18(5):529–36. https://doi.org/10.1038/nsmb.2019
  53. 48. Westra ER, Erp PBGV, Kunne T, Wong SP, Staals RHJ, Seegers CLC, et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 2012;46(5):595–605. https://doi.org/10.1016/j.molcel.2012.03.018
  54. 49. Hochstrasser ML, Taylor DW, Bhat P, Guegler CK, Stenberg SH, Nogales E, et al. CasA mediates Cas3-catalysed target degradation during CRISPR RNA-guided interference. Proc Natl Acad Sci USA 2014;111(188):6618–23. https://doi.org/10.1073/pnas.1405079111
  55. 50. Brendel J, Stoll B, Lange SJ, Sharma K, Lenz C, Stachler AE, et al. A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii. J Biol Chem 2014;289(10):7164–77. https://doi.org/10.1074/jbc.M113.508184
  56. 51. Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 2011;30(7):1335–42. https://doi.org/10.
  57. 1038/emboj.2011.41
  58. 52. Jackson RN, Lavin M, Carter J, Wiedenheft B. Fitting CRISPR-associated Cas3 into the helicase family tree. Curr Opin Struct Biol 2014;24:106–14. https://doi.org/10.1016/j.sbi.2014.01.001
  59. 53. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 2014;5(1):e00928-00913. https://doi.org/10.1128/mBio.00928-13
  60. 54. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816–21. https://doi.org/10.1126/science.
  61. 1225829
  62. 55. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6021):819–23. https://doi.org/10.1126/science.1231143
  63. 56. Mali P, Yang L, Esvelt KM, Arch J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339(6121):823–26. https://doi.org/10.1126/science.1232033
  64. 57. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 2009;155(Pt3):733–40. https://doi.org/10.1099/mic.0.023960-0
  65. 58. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 2012;109(39):E2579–86. https://doi.org/10.1073/pnas.1208507109
  66. 59. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 2015;523(7561):481–85. https://doi.org/10.1038/nature14592
  67. 60. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 2018;556(7699):57–63. https://doi.org/10.1038/nature26155
  68. 61. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science 2016;351(6268):84–88. https://doi.org/10.1126/science.aad5227
  69. 62. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 2016;529(7587):490–95. https://doi.org/10.1038/nature
  70. 16526
  71. 63. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 2017;550(7676):407–10. https://doi.org/10.1038/nature24268
  72. 64. Casini A, Olivieri M, Petris G, Montagna C, Reginata G, Maule G, et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat Biotechnol 2018;36(3):265–71. https://doi.org/10.1038/nbt.4066
  73. 65. Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med 2018;24(8):1216–24. https://doi.org/10.1038/s41591-018-0137-0
  74. 66. Kocak DD, Josephs EA, Bhandarkar V, Adkar SS, Kwon JB, Gersbach CA. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat Biotechnol 2019;37(6):657–66. https://doi.org/10.1038/s415
  75. 87-019-0095-1
  76. 67. Deveau H, Barrangou R, Garmeau JE, Labonte J, Fremaux C, Boyaval P, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 2008; 190(4):1390–400. https://doi.org/10.1128/JB.01412-07
  77. 68. Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA 2013;110(39):15644–49. https://doi.org/10.1073/pnas.1313587110
  78. 69. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015;520(7546):186–91. https://doi.org/10.1038/nature14299
  79. 70. Yamada M, Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, et al. Crystal structure of the minimal Cas9 from Campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems. Mol Cell 2017;65(6):1109–21. https://doi.org/10.1016/j.molcel.2017.02.007
  80. 71. Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun 2017;8(14500):1–12. https://doi.org/10.1038/ncomms14500
  81. 72. Burstein D, Harrington LB, Strutt SC, Probst AJ, Anantharaman K, Thomas BC, et al. New CRISPR-Cas systems from uncultivated microbes. Nature 2017;542(7640):237–41. https://doi.org/10.1038/nature21059
  82. 73. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol 2017;35(1):31–34. https://doi.org/10.1038/nbt.3737
  83. 74. Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol 2019;37(3):276–82. https://doi.org/10.1038/s41587-018-0011-0
  84. 75. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315(5819):1709–12. https://doi.org/10.1126/science.1138140
  85. 76. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8(11):2281–308. https://doi.org/10.1038/nprot.2013.143
  86. 77. Liang F, Han M, Romanienko PJ, Jasin M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proceedings of the National Academy of Sciences. 1998;95(9):5172–77. https://doi.org/10.1073/pnas.95.9.5172
  87. 78. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Molecular and cellular biology. 2001;21(1):289–97. https://doi.org/10.1128/MCB.21.1.289-297.2001
  88. 79. Fishman-Lobell J, Rudin N, Haber JE. Two alternative pathways of double-strand break repair are kinetically separable and independently modulated. Molecular and cellular biology. 1992 Mar 1;12(3):1292–303. https://doi.org/10.1128/mcb.12.3.1292-303.1992
  89. 80. Smurnyy Y, Cai M, Wu H, McWhinnie E, Tallarico JA, Yang Y, et al. DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells. Nat Chem Biol 2014;10(8):623–25. https://doi.org/10.1038/nchembio.1550

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