基因组编辑技术及其安全管理

doi:10.3864/j.issn.0578-1752.2017.08.001

摘要/Abstract

摘要： 基因组编辑技术利用核酸酶对生物体内的DNA双链进行断裂，并以非同源末端连接或同源重组的方式对基因组DNA特定位点进行突变、缺失或者基因的插入与替换。锌指核酸酶、转录激活因子样效应物核酸酶、成簇规律间隔短回文重复序列是目前基因组编辑技术应用中的3种关键核酸酶。基因组编辑技术已在植物基因功能、育种等领域广泛应用，特别是基于成簇规律间隔短回文重复序列的基因编辑技术CRISPR-Cas9。具有优良性状的基因组编辑大豆、玉米等产品已逐步从实验室走向田间，基因组编辑作物展现了较传统转基因作物更为优越的应用前景。本文简要概述了主要使用的3种基因组编辑技术及其原理。对这些技术的优缺点进行了分析，并依据物种分类梳理了利用上述3种技术在动物、植物中突变体建立、基因功能研究、分子育种等方面的研究进展。同时，针对基因编辑产物的产业化应用前景，讨论了基因编辑技术及其产品较传统转基因技术产品的优势，分析了基因编辑技术及其产品可能因脱靶效应而引发的生物安全风险,介绍了美国、欧盟等国家对基因编辑技术及其产品安全管理和商业化应用的政策。文章结合中国现行法规对转基因生物的定义及安全评价（实质等同、个案分析）原则，讨论了基因编辑技术及其产品的安全管理，初步提出了基于传统转基因生物安全评价框架的基因编辑产品的安全评价和管理思路。针对基因编辑产品需要按照个案原则进行评价和管理，安全评价重点开展分子特征及食用安全评价；同时需要针对基因编辑技术的特点建立更加有效、特异的检测新方法，实现对基因编辑产品的有效监测，以促进基因组编辑产品的商业化应用。

关键词: 基因组编辑, 转基因生物, 安全监管

Abstract: Genome editing technologies using sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites, and the DSBs can be repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways with the help of artificially engineered nucleases, which can be employed to achieve targeted genome modifications such as gene mutation, gene insertion, gene replacement or chromosome rearrangement. There are three major artificially engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) systems. The genome editing techniques have been widely used in the fields of plant gene function research, molecular breeding, and etc. The genome editing crops (GEC) show promising application prospects compared with traditional genetically modified organism (GMO). The GECs with good traits have been gradually transferred from labs to fields. Herein, the principles, characters, and applications of these three mainly used techniques in animal and plants genome editing have been described. The advantages and disadvantages compared with conventional transgenic technique were also discussed, including the safety came from the off-target effects. The management regulations of GECs of different countries in global area were elaborated. Finally, the safety management of GECs and their products in China were discussed combined with Chinese current regulations on the safety management of GMOs, so as to facilitate the development and commercialization of GECs and their products.

Key words: genome editing, genetically modified organisms, safety management

沈平，章秋艳，杨立桃，张丽，李文龙，梁晋刚，李夏莹，王颢潜，沈晓玲，宋贵文. 基因组编辑技术及其安全管理[J]. 中国农业科学, 2017, 50(8): 1361-1369.

SHEN Ping, ZHANG QiuYan, YANG LiTao, ZHANG Li, LI WenLong, LIANG JinGang, LI XiaYing, WANG HaoQian, SHEN XiaoLing, SONG GuiWen. The Safety Management of Genome Editing Technology[J]. Scientia Agricultura Sinica, 2017, 50(8): 1361-1369.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.08.001

https://www.chinaagrisci.com/CN/Y2017/V50/I8/1361

参考文献

[1] PAPAIOANNOU I, SIMONS J P, OWEN J S. Oligonucleotide- directed gene-editing technology: mechanisms and future prospects. Expert Opinion on Biological Therapy, 2012, 12(3): 329-342.

[2] PABO C O, PEISACH E, Grant R A. Design and selection of novel Cys2His2 Zinc finger proteins. Annual Review of Biochemistry, 2001, 70: 313-340.

[3] LI T, HUANG S, ZHAO X, WRIGHT D A, CARPENTER S, SPALDING M H, WEEKS D P, YANG B. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Research, 2011, 39(14): 6315-6325.

[4] 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. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.

[5] GAO F, SHEN X Z, JIANG F, WU Y, HAN C. DNA-guided genome editing using the Natrono bacteriumgregoryi Argonaute. Nature Biotechnology, 2016, 34(7): 768-773.

[6] XU S, CAO S, ZOU B, YUE Y, GU C, CHEN X, WANG P, DONG X, XIANG Z, LI K, ZHU M, ZHAO Q, ZHOU G. An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease. Genome Biology, 2016, 17(1): 186.

[7] SINGH A, CHAKRABORTY D, MAITI S. CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering. Chemical Society Reviews, 2016, 45(24): 6666-6684.

[8] JO Y I, KIM H, RAMAKRISHNA S. Recent developments and clinical studies utilizing engineered zinc finger nuclease technology. Cellular and Molecular Life Sciences, 2015, 72(20): 3819-3830.

[9] SPRINK T, METJE J, HARTUNG F. Plant genome editing by novel tools: TALEN and other sequence specific nucleases. Current Opinion in Biotechnology, 2015, 32: 47-53.

[10] CEASAR S A, RAJAN V, PRYKHOZHIJ S V, BERMAN J N, IGNACIMUTHU S. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9. Biochimica et Biophysica Acta, 2016, 1863(9): 2333-2344.

[11] 单奇伟, 高彩霞. 植物基因组编辑及衍生技术最新研究进展. 遗传, 2015, 37(10): 953-973.

SHAN Q W, GAO C X. Research progress of genome editing and derivative technologies in plants. Hereditas, 2015, 37(10): 953-973. (in Chinese)

[12] 周想春, 邢永忠. 基因组编辑技术在植物基因功能鉴定及作物育种中的应用. 遗传, 2016, 38(3): 227-242.

ZHOU X C, XING Y Z. The application of genome editing in identification of plant gene function and crop breeding. Hereditas, 2016, 38(3): 227-242. (in Chinese)

[13] 幸宇云, 杨强, 任军. CRISPR/Cas9基因组编辑技术在农业动物中的应用. 遗传, 2016, 38(3): 217-226.

XING Y Y, YANG Q, REN J. Application of CRISPR/Cas9 mediated genome editing in farm animals. Hereditas, 2016, 38(3): 217-226. (in Chinese)

[14] 王干诚, 马明, 叶延帧, 席建忠. 基于CRISPR/Cas9系统高通量筛选研究功能基因. 遗传, 2016, 38(5): 391-401.

WANG G C, MING M, YE Y Z, XI J Z. High-throughput functional screening using CRISPR/Cas9 system. Hereditas, 2016, 38(5): 391-401. (in Chinese)

[15] CHEN Y Y. Efficient gene editing in primary human T cells. Trends in Immunology, 2015, 36(11): 667-669.

[16] WHITELAW C B, SHEETS T P, LILLICO S G, TELUGU B P. Engineering large animal models of human disease. Journal of Pathology, 2016, 238(2): 247-256.

[17] WON M, RO H, DAWID I B. Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(40): 12426-12431.

[18] SOLIN S L, SHIVE H R, WOOLARD K D, ESSNER J J, MCGRAIL M. Rapid tumor induction in zebrafish by TALEN-mediated somatic inactivation of the retinoblastoma1 tumor suppressor rb1. Scientific Reports, 2015, 5: 13745.

[19] HENDRIKS W T, JIANG X, DAHERON L, COWAN C A. TALEN- and CRISPR/Cas9-mediated gene editing in human pluripotent stem cells using lipid-based transfection. Current Protocols in Stem Cell Biology, 2015, 34: 5B.3.1-5B.3.25.

[20] LEE H B, SEBO Z L, PENG Y, GUO Y. An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster. Cellular Logistics, 2015, 5(1): e1023423.

[21] DAIMON T, UCHIBORI M, NAKAO H, SEZUTSU H, SHINODA T. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(31): 4226-4235.

[22] BUDHAGATAPALLI N, RUTTEN T, GURUSHIDZE M, KUMLEHN J, HENSEL G. Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double-strand breaks in barley. G3 (Bethesda), 2015, 5(9): 1857-1863.

[23] RANI R, YADAV P, BARBADIKAR K M, BALIYAN N, MALHOTRA E V, SINGH B K, KUMAR A, SINGH D. CRISPR/ Cas9: a promising way to exploit genetic variation in plants. Biotechnology Letters, 2016, 38(12): 1991-2006.

[24] XU R, WEI P, YANG J. Use of CRISPR/Cas genome editing technology for targeted mutagenesis in rice. Methods in Molecular Biology, 2017, 1498: 33-40.

[25] LI J, MENG X, ZONG Y, CHEN K, ZHANG H, LIU J, LI J, GAO C. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nature Plants, 2016. 2: 16139.

[26] MA X, LIU Y G. Crispr/cas9-based multiplex genome editing in monocot and dicot plants. Current Protocols in Molecular Biology, 2016, 115: 31.6.1-31.6.21.

[27] SCHIML S, FAUSER F, PUCHTA H. Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(26): 7266-7271.

[28] TANG X, ZHENG X, QI Y, ZHANG D, CHENG Y, TANG A, VOYTAS D F, ZHANG Y. A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Molecular Plant, 2016, 9(7):1088-1091.

[29] PAN C, YE L, QIN L, LIU X, HE Y, WANG J, CHEN L, LU G. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports, 2016, 6: 24765.

[30] LI M, LI X, ZHOU Z, WU P, FANG M, PAN X, LIN Q, LUO W, WU G, LI H. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science, 2016, 7: 377.

[31] YEE J K. Off-target effects of engineered nucleases. Febs Journal, 2016, 283(17): 3239-3248.

[32] SHUKLA V K, DOYON Y, MILLER J C, DEKELVER R C, MOEHLE E A, WORDEN S E, MITCHELL J C, ARNOLD N L, GOPALAN S, MENG X, CHOI V M, ROCK J M, WU Y Y, KATIBAH G E, ZHIFANG G, MCCASKILL D, SIMPSON M A, BLAKESLEE B, GREENWALT S A, BUTLER H J, HINKLEY S J, ZHANG L, REBAR E J, GREGORY P D, URNOV F D. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 2009, 459(7245): 437-441.

[33] CAI C Q, DOYON Y, AINLEY W M, MILLER J C, DEKELVER R C, MOEHLE E A, ROCK J M, LEE Y L, GARRISON R, SCHULENBERG L, BLUE R, WORDEN A, BAKER L, FARAJI F, ZHANG L, HOLMES M C, REBAR E J, COLLINGWOOD T N, RUBIN-WILSON B, GREGORY P D, URNOV F D, PETOLINO J F. Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Molecular Biology, 2009, 69(6): 699-709.

[34] LI T, LIU B, SPALDING M H, WEEKS D P, YANG B. High efficiency TALEN-based gene editing produces disease resistant rice. Nature Biotechnology, 2012, 30(5): 390-392.

[35] JONES H D. Regulatory uncertainty over genome editing. Nature Plants, 2015, 1: 14011.

[36] LEDFORD H. US rethinks crop regulation. Nature, 2016, 532: 158-159.

[37] WALTZ E. Gene-edited CRISPR mushroom escapes US regulation. Nature, 2016,532, 293.

[38] SHAN Q W, ZHANG Y, CHEN K L, ZHANG K, GAO C X. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnology Journal, 2015, 13(6): 791-800.

[39] SHAN Q W, WANG YP, LI J, ZHANG Y, CHEN K L, LIANG Z, ZHANG K, LIU J X, XI J J, QIU J L, GAO C X. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology, 2013, 31(8): 686-688.

[40] WANG Y P, CHENG X, SHAN Q W, ZHANG Y, LIU J X, GAO C X, QIU J L. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 2014, 32(9): 947-951.

[41] YU S, LUO J, SONG Z, DING F, DAI Y, LI N. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Research, 2011, 21(21): 1638-1640.

[42] QIAN L, TANG M, YANG J, WANG Q, CAI C, JIANG S, LI H, JIANG K, GAO P, MA D, CHEN Y, AN X, LI K, CUI W. Targeted mutations in myostatin by zinc-finger nucleases result in double- muscled phenotype in Meishan pigs. Scientific Reports, 2015, 5: 14435.

[43] LIU X, WANG Y, GUO W, CHANG B, LIU J, GUO Z, QUAN F, ZHANG Y. Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows. Nature Communication, 2013, 4: 2565.

[44] LIU X, WANG Y, TIAN Y, YU Y, GAO M, HU G, SU F, PAN S, LUO Y, GUO Z, QUAN F, ZHANG Y. Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases. Proceedings of the Royal Society, 2014, 281: 20133368.

[45] WU H, WANG Y, ZHANG Y, YANG M, LV J, LIU J, ZHANG Y. TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(13): 1530-1539.

[46] LUO Y, WANG Y, LIU J, CUI C, WU Y, LAN H CHEN Q, LIU X, QUAN F, GUO Z, ZHANG Y. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands. Scientific Reports, 2016, 6: 20657.

[47] LI X, YANG Y, BU L, GUO X, TANG C, SONG J, FAN N, ZHAO B, OUYANG Z, LIU Z, ZHAO Y, YI X, QUAN L, LIU S, YANG Z, OUYANG H, CHEN Y E, WANG Z, LAI L. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Research, 2014, 24(4): 501-504.

[48] RUAN J, LI H, XU K, WU T, WEI J, ZHOU R, LIU Z, MU Y, YANG S, OUYANG H, CHEN-TSAI R Y, LI K. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific Reports, 2015, 5: 14253.

[49] HAI T, TENG F, GUO R, LI W, ZHOU Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research, 2014, 24(3): 372-375.

[50] ZHOU X, XIN J, FAN N, ZOU Q, HUANG J, OUYANG Z, ZHAO Y, ZHAO B, LIU Z, LAI S, YI X, GUO L, ESTEBAN MA, ZENG Y, YANG H, LAI L. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 2015, 72(6): 1175-1184.

[51] YAO J, HUANG J, HAI T, WANG X, QIN G, ZHANG H, WU R, CAO C, XI J J, YUAN Z, ZHAO J. Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs. Scientific Reports, 2014, 4: 6926.

[52] WANG X, CAO C, HUANG J, YAO J, HAI T, ZHENG Q, WANG X, ZHANG H, QIN G, CHENG J, WANG Y, YUAN Z, ZHOU Q, WANG H, ZHAO J. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system. Scientific Reports, 2016, 6: 20620.

[53] PENG J, WANG Y, JIANG J, ZHOU X, SONG L, WANG L, DING C, QIN J, LIU L, WANG W, LIU J, HUANG X, WEI H, ZHANG P. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Scientific Reports, 2015, 5: 16705.

[54] YANG Y, WANG K, WU H, JIN Q, RUAN D, OUYANG Z, ZHAO B, LIU Z, ZHAO Y, ZHANG Q, FAN N, LIU Q, GUO S, BU L, FAN Y, SUN X, LI X, LAI L. Genetically humanized pigs exclusively expressing human insulin are generated through custom endonuclease- mediated seamless engineering. Journal of Molecular Cell Biology, 2016, 8(2): 174-177.

[55] WHELAN A I, LEMA M A. Regulatory framework for gene editing and other new breeding techniques (NBTs) in Argentina. GM Crops Food, 2015, 6(4): 253-265.

[56] HUANG S, WEIGEL D, BEACHY RN, LI J. A proposed regulatory framework for genome-edited crops. Nature Genetics, 2016, 48(2): 109-111.

[57] JONES H D. Future of breeding by genome editing is in the hands of regulators. GM Crops Food, 2015, 6(4): 223-232.