Plant Biotechnology Reports

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Targeted genome engineering via zinc finger nucleases

  • Kim, Seok-Joong (ToolGen, Inc., Biotechnology Incubating Center, Seoul National University) ;
  • Kim, Jin-Soo (Department of Chemistry, Seoul National University)
  • Received : 2010.12.07
  • Accepted : 2010.12.09
  • Published : 2011.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.

Keywords

References

  1. Agarwal S, Tafel AA, Kanaar R (2006) DNA double-strand break repair and chromosome translocations. DNA Repair (Amst) 5:1075-1081 https://doi.org/10.1016/j.dnarep.2006.05.029
  2. Bae KH, Kwon YD, Shin HC, Hwang MS, Ryu EH, Park KS, Yang HY, Lee DK, Lee Y, Park J, Kwon HS, Kim HW, Yeh BI, Lee HW, Sohn SH, Yoon J, Seol W, Kim JS (2003) Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat Biotechnol 21:275-280 https://doi.org/10.1038/nbt796
  3. Beumer K, Bhattacharyya G, Bibikova M, Trautman JK, Carroll D (2006) Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172:2391-2403
  4. Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci USA 105:19821-19826 https://doi.org/10.1073/pnas.0810475105
  5. Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161:1169-1175
  6. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764 https://doi.org/10.1126/science.1079512
  7. Brunet E, Simsek D, Tomishima M, DeKelver R, Choi VM, Gregory P, Urnov F, Weinstock DM, Jasin M (2009) Chromosomal translocations induced at specified loci in human stem cells. Proc Natl Acad Sci USA 106:10620-10625 https://doi.org/10.1073/pnas.0902076106
  8. Cai CQ, Doyon Y, Ainley WM, Miller JC, Dekelver RC, Moehle EA, Rock JM, Lee YL, Garrison R, Schulenberg L, Blue R, Worden A, Baker L, Faraji F, Zhang L, Holmes MC, Rebar EJ, Collingwood TN, Rubin-Wilson B, Gregory PD, Urnov FD, Petolino JF (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol 69:699-709 https://doi.org/10.1007/s11103-008-9449-7
  9. Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6:507-512
  10. Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L, Cui X (2010) Targeted genome modification in mice using zincfinger nucleases. Genetics 186:451-459 https://doi.org/10.1534/genetics.110.117002
  11. Carroll D, Morton JJ, Beumer KJ, Segal DJ (2006) Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc 1:1329-1341 https://doi.org/10.1038/nprot.2006.231
  12. Carroll D, Beumer KJ, Morton JJ, Bozas A, Trautman JK (2008) Gene targeting in Drosophila and Caenorhabditis elegans with zinc-finger nucleases. Methods Mol Biol 435:63-77
  13. Chandrasegaran S, Smith J (1999) Chimeric restriction enzymes: what is next? Biol Chem 380:841-848
  14. Cost GJ, Freyvert Y, Vafiadis A, Santiago Y, Miller JC, Rebar E, Collingwood TN, Snowden A, Gregory PD (2010) BAK and BAX deletion using zinc-finger nucleases yields apoptosisresistant CHO cells. Biotechnol Bioeng 105:330-340 https://doi.org/10.1002/bit.22541
  15. de Pater S, Neuteboom LW, Pinas JE, Hooykaas PJ, van der Zaal BJ (2009) ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnol J 7:821-835 https://doi.org/10.1111/j.1467-7652.2009.00446.x
  16. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26:702-708 https://doi.org/10.1038/nbt1409
  17. Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S (2005) Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res 33:5978-5990 https://doi.org/10.1093/nar/gki912
  18. Foley JE, Yeh JR, Maeder ML, Reyon D, Sander JD, Peterson RT, Joung JK (2009) Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN). PLoS One 4:e4348 https://doi.org/10.1371/journal.pone.0004348
  19. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Menoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433 https://doi.org/10.1126/science.1172447
  20. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851-857 https://doi.org/10.1038/nbt.1562
  21. Jamieson AC, Miller JC, Pabo CO (2003) Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2:361-368 https://doi.org/10.1038/nrd1087
  22. Johnson RD, Jasin M (2001) Double-strand-break-induced homologous recombination in mammalian cells. Biochem Soc Trans 29:196-201 https://doi.org/10.1042/BST0290196
  23. Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93:1156-1160 https://doi.org/10.1073/pnas.93.3.1156
  24. Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS (2009) Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res 19:1279-1288 https://doi.org/10.1101/gr.089417.108
  25. Kim JS, Lee HJ, Carroll D (2010a) Genome editing with modularly assembled zinc-finger nucleases. Nat Methods 7:91 (author reply 91-92)
  26. Kim S, Lee MJ, Kim H, Kang M, Kim JS (2010b) Pre-assembled zinc finger arrays for rapid construction of zinc finger nucleases. Nat Methods (in press)
  27. Klug A (2010) The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev Biochem 79:213-231 https://doi.org/10.1146/annurev-biochem-010909-095056
  28. Lee DK, Seol W, Kim JS (2003) Custom DNA-binding proteins and artificial transcription factors. Curr Top Med Chem 3:645-657 https://doi.org/10.2174/1568026033452384
  29. Lee HJ, Kim E, Kim JS (2010) Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res 20:81-89 https://doi.org/10.1101/gr.099747.109
  30. Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181-211 https://doi.org/10.1146/annurev.biochem.052308.093131
  31. Liu PQ, Chan EM, Cost GJ, Zhang L, Wang J, Miller JC, Guschin DY, Reik A, Holmes MC, Mott JE, Collingwood TN, Gregory PD (2010) Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases. Biotechnol Bioeng 106:97-105
  32. Lloyd A, Plaisier CL, Carroll D, Drews GN (2005) Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci USA 102:2232-2237 https://doi.org/10.1073/pnas.0409339102
  33. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, Holmes MC, Naldini L (2007) Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 25:1298-1306 https://doi.org/10.1038/nbt1353
  34. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, Unger-Wallace E, Sander JD, Muller-Lerch F, Fu F, Pearlberg J, Gobel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, McCray PB Jr, Cathomen T, Voytas DF, Joung JK (2008) Rapid ''open-source'' engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31:294-301 https://doi.org/10.1016/j.molcel.2008.06.016
  35. Maeder ML, Thibodeau-Beganny S, Sander JD, Voytas DF, Joung JK (2009) Oligomerized pool engineering (OPEN): an ''opensource'' protocol for making customized zinc-finger arrays. Nat Protoc 4:1471-1501 https://doi.org/10.1038/nprot.2009.98
  36. Mandell JG, Barbas CF 3rd (2006) Zinc finger tools: custom DNAbinding domains for transcription factors and nucleases. Nucleic Acids Res 34:W516-W523 https://doi.org/10.1093/nar/gkl209
  37. Marton I, Zuker A, Shklarman E, Zeevi V, Tovkach A, Roffe S, Ovadis M, Tzfira T, Vainstein A (2010) Nontransgenic genome modification in plant cells. Plant Physiol 154:1079-1087 https://doi.org/10.1104/pp.110.164806
  38. Mashimo T, Takizawa A, Voigt B, Yoshimi K, Hiai H, Kuramoto T, Serikawa T (2010) Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. PLoS One 5:e8870 https://doi.org/10.1371/journal.pone.0008870
  39. Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol 26:695-701 https://doi.org/10.1038/nbt1398
  40. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778-785 https://doi.org/10.1038/nbt1319
  41. Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Gregory PD, Urnov FD, Holmes MC (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci USA 104:3055-3060 https://doi.org/10.1073/pnas.0611478104
  42. Moore M, Klug A, Choo Y (2001) Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proc Natl Acad Sci USA 98:1437-1441 https://doi.org/10.1073/pnas.98.4.1437
  43. Morton J, Davis MW, Jorgensen EM, Carroll D (2006) Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Proc Natl Acad Sci USA 103:16370-16375 https://doi.org/10.1073/pnas.0605633103
  44. Ochiai H, Fujita K, Suzuki K, Nishikawa M, Shibata T, Sakamoto N, Yamamoto T (2010) Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15:875-885
  45. Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J, Miller JC, Holmes MC, Gregory PD, Urnov FD, Cost GJ (2010) Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res 38:e152 https://doi.org/10.1093/nar/gkq512
  46. Osakabe K, Osakabe Y, Toki S (2010) Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases. Proc Natl Acad Sci USA 107:12034-12039 https://doi.org/10.1073/pnas.1000234107
  47. Pabo CO, Peisach E, Grant RA (2001) Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem 70:313-340 https://doi.org/10.1146/annurev.biochem.70.1.313
  48. Park KS, Lee DK, Lee H, Lee Y, Jang YS, Kim YH, Yang HY, Lee SI, Seol W, Kim JS (2003) Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors. Nat Biotechnol 21:1208-1214 https://doi.org/10.1038/nbt868
  49. Park KS, Jang YS, Lee H, Kim JS (2005) Phenotypic alteration and target gene identification using combinatorial libraries of zinc finger proteins in prokaryotic cells. J Bacteriol 187:5496-5499 https://doi.org/10.1128/JB.187.15.5496-5499.2005
  50. Petolino JF, Worden A, Curlee K, Connell J, Strange Moynahan TL, Larsen C, Russell S (2010) Zinc finger nuclease-mediated transgene deletion. Plant Mol Biol 73:617-628 https://doi.org/10.1007/s11103-010-9641-4
  51. Porteus MH, Carroll D (2005) Gene targeting using zinc finger nucleases. Nat Biotechnol 23:967-973 https://doi.org/10.1038/nbt1125
  52. Puchta H, Dujon B, Hohn B (1993) Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Res 21:5034-5040 https://doi.org/10.1093/nar/21.22.5034
  53. Ramirez CL, Foley JE, Wright DA, Muller-Lerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA, Cathomen T, Voytas DF, Joung JK (2008) Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5:374-375 https://doi.org/10.1038/nmeth0508-374
  54. Remy S, Tesson L, Menoret S, Usal C, Scharenberg AM, Anegon I (2010) Zinc-finger nucleases: a powerful tool for genetic engineering of animals. Transgenic Res 19:363-371 https://doi.org/10.1007/s11248-009-9323-7
  55. San Filippo J, Sung P, Klein H (2008) Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77:229-257 https://doi.org/10.1146/annurev.biochem.77.061306.125255
  56. Sander JD, Zaback P, Joung JK, Voytas DF, Dobbs D (2007) Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res 35:W599-W605 https://doi.org/10.1093/nar/gkm349
  57. Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D (2010) ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res 38(Suppl):W462-W468 https://doi.org/10.1093/nar/gkq319
  58. Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci USA 105:5809-5814 https://doi.org/10.1073/pnas.0800940105
  59. Segal DJ, Dreier B, Beerli RR, Barbas CF 3rd (1999) Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci USA 96:2758-2763 https://doi.org/10.1073/pnas.96.6.2758
  60. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise genome modification in the crop species Zea mays using zincfinger nucleases. Nature 459:437-441 https://doi.org/10.1038/nature07992
  61. Siekmann AF, Standley C, Fogarty KE, Wolfe SA, Lawson ND (2009) Chemokine signaling guides regional patterning of the first embryonic artery. Genes Dev 23:2272-2277 https://doi.org/10.1101/gad.1813509
  62. Smithies O (2001) Forty years with homologous recombination. Nat Med 7:1083-1086 https://doi.org/10.1038/nm1001-1083
  63. Sollu C, Pars K, Cornu TI, Thibodeau-Beganny S, Maeder ML, Joung JK, Heilbronn R, Cathomen T (2010) Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion. Nucleic Acids Res. doi:10.1093/nar/gkq720
  64. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25:786-793 https://doi.org/10.1038/nbt1317
  65. Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, Carroll D, Tamura T, Zurovec M (2010) Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol 40:759-765 https://doi.org/10.1016/j.ibmb.2010.07.012
  66. Tovkach A, Zeevi V, Tzfira T (2009) A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells. Plant J 57:747-757 https://doi.org/10.1111/j.1365-313X.2008.03718.x
  67. Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459:442-445 https://doi.org/10.1038/nature07845
  68. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646-651 https://doi.org/10.1038/nature03556
  69. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636-646 https://doi.org/10.1038/nrg2842
  70. Vasquez KM, Marburger K, Intody Z, Wilson JH (2001) Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci USA 98:8403-8410 https://doi.org/10.1073/pnas.111009698
  71. Wolfe SA, Nekludova L, Pabo CO (2000) DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29:183-212 https://doi.org/10.1146/annurev.biophys.29.1.183
  72. Wright DA, Townsend JA, Winfrey RJ Jr, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) Highfrequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693-705 https://doi.org/10.1111/j.1365-313X.2005.02551.x
  73. Wright DA, Thibodeau-Beganny S, Sander JD, Winfrey RJ, Hirsh AS, Eichtinger M, Fu F, Porteus MH, Dobbs D, Voytas DF, Joung JK (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc 1:1637-1652 https://doi.org/10.1038/nprot.2006.259
  74. Wyman C, Kanaar R (2006) DNA double-strand break repair: all's well that ends well. Annu Rev Genet 40:363-383 https://doi.org/10.1146/annurev.genet.40.110405.090451
  75. Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D, Christian M, Li X, Pierick CJ, Dobbs D, Peterson T, Joung JK, Voytas DF (2010) High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci USA 107:12028-12033 https://doi.org/10.1073/pnas.0914991107
  76. Zou J, Maeder ML, Mali P, Pruett-Miller SM, Thibodeau-Beganny S, Chou BK, Chen G, Ye Z, Park IH, Daley GQ, Porteus MH, Joung JK, Cheng L (2009) Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5:97-110 https://doi.org/10.1016/j.stem.2009.05.023

Cited by

  1. LAHEDES: the LAGLIDADG homing endonuclease database and engineering server vol.40, pp.1, 2011, https://doi.org/10.1093/nar/gks365
  2. Homing endonucleases: DNA scissors on a mission vol.55, pp.8, 2011, https://doi.org/10.1139/g2012-049
  3. Transgenic technologies for enhanced molecular breeding of white clover (Trifolium repens L.) vol.64, pp.1, 2011, https://doi.org/10.1071/cp12184
  4. Engineering nucleases for gene targeting: safety and regulatory considerations vol.31, pp.1, 2014, https://doi.org/10.1016/j.nbt.2013.07.001
  5. Emerging applications of genome-editing technology to examine functionality of GWAS-associated variants for complex traits vol.50, pp.7, 2011, https://doi.org/10.1152/physiolgenomics.00028.2018
  6. CRISPR and Target-Specific DNA Endonucleases for Efficient DNA Knock-in in Eukaryotic Genomes vol.41, pp.11, 2011, https://doi.org/10.14348/molcells.2018.0408
  7. Universal CARs, universal T cells, and universal CAR T cells vol.11, pp.1, 2018, https://doi.org/10.1186/s13045-018-0677-2
  8. Functional genomic approaches to improve crop plant heat stress tolerance vol.8, pp.None, 2019, https://doi.org/10.12688/f1000research.19840.1
  9. Genome Editing Tools: Need of the Current Era vol.9, pp.3, 2011, https://doi.org/10.4236/ajmb.2019.93008