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유전자교정작물 내 비의도적 돌연변이의 안전성 논란에 관한 과학적 고찰

Scientific considerations for the biosafety of the off-target effects of gene editing in crops

  • 이신우 (국립경남과학기술대학교 생명과학대학 농학.한약자원학부) ;
  • 김윤희 (국립경상대학교 사범대학 생물교육과(농업생명과학연구원))
  • Lee, Shin-Woo (Department of Agronomy & Medicinal Plant Resources, Gyeongnam National University of Science & Technology) ;
  • Kim, Yun-Hee (Department of Biology Education, College of Education, IALS, Gyeongsang National University)
  • 투고 : 2020.06.18
  • 심사 : 2020.08.03
  • 발행 : 2020.09.30

초록

최근, 유전자교정 작물의 상업화 승인 건수가 급속하게 늘어나고 있으며, 국내에서도 유전자교정 작물의 개발에 대한 집중적인 투자를 통하여 국제경쟁력을 높이기 위하여 노력하고 있다. 그러나 기존의 유전자변형작물의 상업화 과정에서 끊임없이 제기되어온 인체 및 환경에 대한 잠재적인 위해성 논란이 유전자교정 작물에 대하여서도 제기되고 있다. 특히, 비의도적 돌연변이(off-target)가 가장 큰 논란의 중심이 되고 있다. 따라서 본 리뷰는 식물이 내포하고 있는 장점인 배수체, 체세포 돌연변이 그리고 자연 상태에서 아그로박테리아의 T-DNA 단편의 수평 전이로 창출된 자연적인 유전자변형작물과 기존에 상업화가 승인된 유전자변형작물 이벤트들의 게놈 내 비의도적 돌연변이 사례 등을 검토한 결과 유전자교정 작물에서 나타나는 대부분의 비의도적 돌연변이는 인체 및 환경에 미칠 수 있는 위해성을 우려할 만한 수준이 아니라고 할 수 있었다. 이에, 유전자교정 작물의 안전성 평가를 위하여 새로운 규정을 제정할 필요가 없으며 기존의 유전자변형작물의 안전 관리규정을 일부 "용어의 정의" 등만 개정하여 적용하면 충분할 것으로 사료 되었다.

The number of commercially approved gene-edited crops is gradually increasing, and in South Korea, it has led to intense investment in gene-edited crop development to increase international competitiveness. However, as with genetically modified crops, the safety of gene-edited crops regarding unexpected risks for humans and the environment is subject to an ongoing debate. In particular, unintentional "off-target effects" have become the center of controversy. In this review, we discuss typical plant characteristics (including somatic variation and ploidy), the extent of various off-target effects in genetically modified crops generated via horizontal transfer in nature, and the off-target effects in commercial genetically modified crops. We conclude that most off-target effects possibly occurring in gene-edited crops are not expected to be critically harmful to humans or the environment. Therefore, existing regulation for genetically modified crops should be enough for the risk assessment of gene-edited crops.

키워드

참고문헌

  1. Agapito-Tenfen AZ, Okoli AS, Bernstein MJ, Odd-Gunnar Wikmark O-G, Myhr AI (2018) Revisiting risk governance of GM plants: the need to consider new and emerging geneediting techniques. Front Plant Sci 9:1874-1890 https://doi.org/10.3389/fpls.2018.01874
  2. Aoki S, Kawaoka A, Sekine M, Ichikawa T, Fujita T, Shinmyo A (1994) Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii. Mol Gen Genet 243:706-710 https://doi.org/10.1007/BF00279581
  3. Aoki S, Syono K (1999) Horizontal gene transfer and mutation: ngrol genes in the genome of Nicotiana glauca. Proc Natl Acad Sci USA 96:13229-13234 https://doi.org/10.1073/pnas.96.23.13229
  4. Aoki S (2004) Resurrection of an ancestral gene: Functional and evolutionary analyses of the Ngrol genes transferred from Agrobacterium to Nicotiana. J Plant Res 117:329-337 https://doi.org/10.1007/s10265-004-0163-5
  5. Carroll D, Van Eenennaam AL, Taylor JF, Seger J, Voytas DF (2016) Regulate genome-edited products, not genome editing itself. Nat Biotechnol 34:477-479 https://doi.org/10.1038/nbt.3566
  6. Chen K, Dorlhac de Borne F, Szegedi E, Otten L (2014) Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T-DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana. Plant J 80:669-682 https://doi.org/10.1111/tpj.12661
  7. Chen K, Borne FD, Julio E, Obszynski J, Pale P, Otten L (2016) Rootspecific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum. Plant J 87:258-269 https://doi.org/10.1111/tpj.13196
  8. Chen K, Otten L (2017) Natural Agrobacterium transformants: Recent results and some theoretical considerations. Front Plant Sci 8:1600-1616 https://doi.org/10.3389/fpls.2017.01600
  9. Costantino P, Capone I, Cardarelli M, De Paolis A, Mauro ML, Trovato M (1994) Bacterial plant oncogenes: the rol genes' saga. Genetica 94:203-211 https://doi.org/10.1007/BF01443434
  10. Endo M, Mikami M, Toki S (2014) Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol 56:41-47 https://doi.org/10.1093/pcp/pcu154
  11. Feng Z, Mao Y. Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z, Zheng R, Yang L (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632-4637 https://doi.org/10.1073/pnas.1400822111
  12. Furner IJ, et al. (1986) An Agrobacterium transformation in the evolution of the genus Nicotiana. Nature 319:422-427 https://doi.org/10.1038/319422a0
  13. Hajiahmadi Z, Shirzadian-Khorramabad R, Kazemzad M, Sohani MM (2019) Enhancement of tomato resistance to Tuta absoluta using a new efficient mesoporous silica nanoparticle-mediated plant transient gene expression approach. Sci Hortic 243:367-375 https://doi.org/10.1016/j.scienta.2018.08.040
  14. Hahn F, Nekrasov V (2019) CRISPR/Cas precision: Do we need to worry about off-targeting in plants? Plant Cell Rep 38:437-441 https://doi.org/10.1007/s00299-018-2355-9
  15. Hajiahmadi Z, Movahedi A, Wei H, Li D, Orooji Y, Ruan H, Zhuge Q (2019) Strategies to increase on-target and reduce off-target effects of the CRISPR/Cas9 system in plants. Int J Mol Sci 20: 3719-3738 https://doi.org/10.3390/ijms20153719
  16. He Y, Wang R, Dai X, Zhao Y (2017) On improving CRISPR for editing plant genes: Ribozyme-mediated guide RNA production and fluorescence-based technology for isolating transgene-free mutants generated by CRISPR. In Progress in Molecular Biology and Translational Science; Elsevier: Amsterdam, The Netherlands, Volume 149, pp. 151-166
  17. Hilscher J, Burstmayr H, Stoger E (2017) Targeted modification of plant genomes for precision crop breeding. Biotechnol J 12:1-4
  18. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827-832 https://doi.org/10.1038/nbt.2647
  19. Intrieri MC, Buiatti M (2001) The horizontal transfer of Agrobacterium rhizogenes genes and the evolution of the genus Nicotiana. Mol Phylogenet Evol 20:100-110 https://doi.org/10.1006/mpev.2001.0927
  20. ISAAA brief 54 (2019) Executive Summary, Global Status of Commercialized Biotech/GM Crops in 2018
  21. Jacobs TB, LaFayette PR, Schmitz JR, Parrott WA (2015) Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol 15:16 https://doi.org/10.1186/s12896-015-0131-2
  22. Jones HD (2015) Future of breeding by genome editing is in the hands of regulators. GM Crops Food 6:223-232 https://doi.org/10.1080/21645698.2015.1134405
  23. Kovacova V, Zluvova J, Janousek B, Talianova M, Vyskot B (2014) The evolutionary fate of the horizontally transferred Agrobacterial mikimopine synthase eene in the genera Nicotiana and Linaria. PLoS ONE 9:e113872 https://doi.org/10.1371/journal.pone.0113872
  24. Kyndt T, Quispea D, Zhaic H, Jarret R, Ghislain M, Liu Q, Gheysen G, Kreuze JF (2015) The genome of cultivated sweetpotato contains Agrobacterium T-DNAs with expressed genes: an example of a naturally transgenic food crop. Proc Natl Acad Sci USA 112:5844-5849 https://doi.org/10.1073/pnas.1419685112
  25. Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron NJ, Uauy C, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Gen Biol 30:258-271
  26. Lee K, Zhang Y, Kleinstiver BP, Guo JA, Aryee MJ, Miller J, Malzahn A, Zarecor S, Lawrence-Dill CJ, Joung JK (2019) Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol J 17:362-372 https://doi.org/10.1111/pbi.12982
  27. Lee SW (2011) Strategies for the development of GM crops in accordance with the environmental risk assessment (I). J Plant Biotechnol 38:1-5 https://doi.org/10.5010/JPB.2011.38.1.001
  28. Lee SW (2018) Strengthening the competitiveness of agricultural biotechnology through practical application of gene editing technology. J Plant Biotechnol 45:135-170
  29. Lee SW (2019) Current status on the modification of the scope for GMO regulation on the gene edited plants with no remnants of inserted foreign DNA fragments. J Plant Biotechnol 46:137-142 https://doi.org/10.5010/JPB.2019.46.3.137
  30. Lemcke K, Schmülling T (1998) Gain of function assays identify non- rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity. Plant J 15:423-433 https://doi.org/10.1046/j.1365-313X.1998.00223.x
  31. Li Z, Liu Z, Xing A, Moon BP, Koellhoffer JP, Huang L, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960-970 https://doi.org/10.1104/pp.15.00783
  32. Li J, Manghwar H, Sun L, Wang P, Wang G, Sheng H, Zhang J, Liu H, Qin L, Rui H, Li B, Lindsey K, Daniell H, Jin S, Zhang X (2019) Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants Plant Biotechol J 17:858-868 https://doi.org/10.1111/pbi.13020
  33. Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261-14266 https://doi.org/10.1038/ncomms14261
  34. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM 92013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833-838 https://doi.org/10.1038/nbt.2675
  35. Matveeva TV, Bogomaz DI, Pavlova OA, Nester EW, Lutova LA (2012) Horizontal gene transfer from genus Agrobacterium to the plant linaria in nature. Mol Plant Microbe Interact 2512:1542-1551
  36. Matveeva TV, Kosachev PA (2013) "Sequences homologous to Agrobacterium rhizogenes rolC In the genome of Linaria acutiloba, "in International Conference on Frontiers of Environment, Energy and Bioscience (ICFEEB 2013). (Lancaster, PA:DES tech Publications, Inc.), 541-546
  37. Matveeva TV, Lutova LA (2014) Horizontal gene transfer from Agrobacterium to plants. Front Plant Sci 5:1-11
  38. Matveeva TV (2018) Agrobacterium-mediated transformation in the evolution of plants. Curr Top Microbiol Immunol 418: 421-441
  39. Mohajjel-Shoja H, Clement B, Perot J, Alioua M, Otten L (2011) Biological activity of the Agrobacterium rhizogenes-derived trolC gene of Nicotiana tabacum and its functional relation to other plast genes. Mol Plant-Microbe Interact 24:44-53 https://doi.org/10.1094/MPMI-06-10-0139
  40. Pavlova OA, Matveeva TV, Lutova LA (2013) Linaria dalmatica genome contains a homologue of rolC gene of Agrobacterium rhizogenes. Eco Genet 11:10-15 https://doi.org/10.17816/ecogen11210-15
  41. Pavlova O, Matveeva T, Lutova L (2014). Genome of Linaria dalmatica contains Agrobacterium rhizogenes RolC gene homolog. Russ J Genet Appl Res 4:461-465 https://doi.org/10.1134/S2079059714050116
  42. Peterson BA, Haak DC, Nishimura MT, Teixeira PJ, James SR, Dangl JL, Nimchuk ZL (2016) Genome-wide assessment of efficiency and specificity in CRISPR/Cas9 mediated multiple site targeting in Arabidopsis. PLoS ONE 16:11, e0162169 https://doi.org/10.1371/journal.pone.0162169
  43. Quispe-Huamanquispe DG, Gheysen G, Kreuze JF (2017) Horizontal gene transfer contributes to plant evolution: The case of Agrobacterium T-DNAs. Front Plant Sci 8:2015-2021 https://doi.org/10.3389/fpls.2017.02015
  44. Quispe-Huamanquis DG, Gheysen G, Yang J, Jarret R, Rossel G, Kreuze JF (2019) The horizontal gene transfer of Agrobacterium T-DNAs into the series Batatas (Genus Ipomoea) genome is not confined to hexaploid sweetpotato. Sci Rep 9:12584-12597 https://doi.org/10.1038/s41598-019-48691-3
  45. Rosati A, Bogani P, Santarlasci A, Buiatti M (2008) Characterisation of 3' transgene insertion site and derived mRNAs in MON810 YieldGard maize. Plant Mol Biol 67:271-281 https://doi.org/10.1007/s11103-008-9315-7
  46. Salomon S, Puchta H (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J 17:6086-6095 https://doi.org/10.1093/emboj/17.20.6086
  47. Shan QY, Wang K, Chen Z, Liang J, Li Y, Zhang K, Zhang J, Liu DFV, Zheng X (2013) Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol Plant 6:1365-1368 https://doi.org/10.1093/mp/sss162
  48. Spano L, Pompon M, Costantino P, van Slogteren GMS, Tempe J (1982) Identificationof T-DNA in the root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855. Plant Mol Biol 1:291-304 https://doi.org/10.1007/BF00027560
  49. Suzuki K, Yamashita I, Tanaka N (2002) Tobacco plants were transformed by Agrobacterium rhizogenes infection during their evolution. Plant J 32:775-787 https://doi.org/10.1046/j.1365-313X.2002.01468.x
  50. Tanaka N (2008) Horizontal gene transfer in Agrobacterium: from Biology to Biotechnology, eds T. Tzfira and V. Citovsky (NewYork, NY: Springer), 623-647
  51. Tang X, Liu G, Zhou J Xu T, Ren Q, You Q, Tian L, Xin X, Zhong Z, Liu B, Zheng X, Zhang D, Malzahn A, Gong Z, Qi Y, Zhang T, Zhang Y (2018) A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biol 19:84-101 https://doi.org/10.1186/s13059-018-1458-5
  52. White FD, Garfinkel J, Huffman GA, Gordon MP, Nester EW (1983) Sequences homologous to Agrobacterium rhizogenes T-DNA in the genomes of uninfected plants. Nature 301:348-350 https://doi.org/10.1038/301348a0
  53. Windels P, Taverniers I, Depicker A, van Bockstaele E, De Loose M (2001) Characterisation of the roundup ready soybean insert. Eur Food Res Technol 213:107-112 https://doi.org/10.1007/s002170100336
  54. Wolt JD, Wang K, Sashital D, Lawrence-Dill CJ (2016) Achieving plant CRISPR targeting that limits off-target effects. Plant Genome 9: 1-8
  55. Woo J W, Kim J, Kwon S I, Corvalan C, Cho S W, Kim H, Kim S G, Kim S T, Choe S, Kim J S (2015) DNA-free genome editing in plants with preassembled CRISPRCas9 ribonucleoproteins. Nat Biotechnol 33(11):1162-1164 https://doi.org/10.1038/nbt.3389
  56. Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6:1975-1983 https://doi.org/10.1093/mp/sst119
  57. Young J, Zastrow-Hayes G, Deschamps S, Svitashev S, Zaremba M, Acharya A, Paulraj S, Peterson-Burch B, Schwartz C, Djukanovic V, Lenderts B, Feigenbutz L, Wang L, Alarcon C, Siksnys V, May G, N. Chilcoat D, Kumar S (2019) CRISPR-Cas9 editing in maize: systematic evaluation of off-target activity and its relevance in crop improvement. Sci Rep 9:6729-6740 https://doi.org/10.1038/s41598-019-43141-6
  58. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu JK (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797-807 https://doi.org/10.1111/pbi.12200
  59. Zhang Q, Xing HL, Wang ZP, Zhang HY, Yang F, Wang XC, Chen QJ (2018) Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention. Plant Mol Biol 96:445-456 https://doi.org/10.1007/s11103-018-0709-x
  60. Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42:10903-10914 https://doi.org/10.1093/nar/gku806