基因组编辑技术应用于作物遗传改良的进展与挑战

doi:10.3864/j.issn.0578-1752.2018.01.001

摘要/Abstract

摘要： 基因组定点编辑（site-specific genome editing）是指在基因组水平上对生物DNA序列进行定点改造的遗传操作技术，其在基因功能解析、动植物遗传改良和新品种培育等方面具有重大的应用价值。基因组定点编辑工作原理是利用序列特异性核酸内切酶（sequence-specific nucleases，SSNs）在基因组靶定位置切割DNA双链，造成DNA双链断裂（DNA double-strand breaks，DSBs），并通过非同源末端连接（non-homologous end joining，NHEJ）或同源重组（homology-directed repair，HDR）的DNA修复途径在基因组特定位点造成靶标基因的碱基插入、缺失或DNA片段替换，从而实现基因组的定点改造。目前，已成功应用于作物遗传改良的SSNs主要包括锌指核酸酶（Zinc finger nucleases，ZFNs）、类转录激活因子效应物核酸酶（transcription activator-like effector nucleases，TALENs）、成簇的规律间隔的短回文重复序列及其相关系统（clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins，CRISPR/Cas system）。从发展态势看，基于CRISPR/Cas系统的基因组编辑技术必将成为作物遗传改良和分子设计育种的核心技术之一。论文简要概述了ZFNs、TALENs和CRISPR/Cas系统这3种基因组编辑的技术背景及工作原理；结合案例重点综述上述3种技术在作物产量、品质、抗病性、抗逆性改良及水稻雄性不育系创制上的研究进展；详细梳理基于CRISPR/Cas的植物基因组单碱基编辑系统和DNA-free植物基因组编辑系统的技术创新和应用；比较分析3种技术的优缺点，并提出基因组编辑技术应用于作物遗传改良的一般原则；介绍了美国和欧盟等对基因编辑技术及其产品安全监管和商业化应用的政策法规，及业界人士对基因编辑作物提出的监管框架协议；最后，针对基因编辑技术自身的技术优势和缺陷，讨论该技术应用于作物遗传改良和分子育种的机遇和挑战。

关键词: 基因组编辑, 作物育种, 遗传改良, TALENs, CRISPR/Cas

Abstract: Site-specific genome editing refers to the genetic manipulation techniques that can precisely modify DNA sequences at the genomic level, which holds a great application value in analysis of gene function, plant genetic improvement or development of new varieties. The general working principle of genome editing is that the sequence-specific nucleases (SSNs) are used to create DNA double-strand breaks (DSBs) at the genomic target sites that will be repaired through non-homologous end joining (NHEJ) and/or homology-directed repair (HDR) pathways, resulting in nucleotide insertion, deletion, or DNA fragment replacement in the targeted gene(s). Nowadays, genome editing technologies including Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/ Cas）system have been successfully applied in genetic improvement of crops. From the perspective of development trend, CRISPR/ Cas system-based genome editing technology will become the core technology in genetic improvement and molecular design breeding of crops. This review starts with brief introduction of the concepts and working principles of ZFNs, TALENs and CRISPR/Cas system, and focus on the recent progress of genome editing-based crop improvement of yield, quality, disease resistance, abiotic stress resistance and rice male sterile line development. Then the authors sorted out the technological innovation and application of CRISPR/Cas-based single-base editing for plant genomes and the DNA-free plant genome editing system in details. After comparing the advantages and disadvantages of different genome editing techniques, some general principles of adoption of genome editing technologies in crop improvement were put forward. Finally, the regulations on genome-edited crops, as well as the opportunities and challenges of genome editing-based crop breeding were discussed.

Key words: genome editing, crop breeding, genetic improvement, TALENs, CRISPR/Cas

王福军，赵开军. 基因组编辑技术应用于作物遗传改良的进展与挑战[J]. 中国农业科学, 2018, 51(1): 1-16.

WANG FuJun, ZHAO KaiJun. Progress and Challenge of Crop Genetic Improvement via Genome Editing[J]. Scientia Agricultura Sinica, 2018, 51(1): 1-16.

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.0578-1752.2018.01.001

https://www.chinaagrisci.com/CN/Y2018/V51/I1/1

参考文献

[1] Voytas D F. Plant genome engineering with sequence-specific nucleases. Annual Review of Plant Biology, 2013, 64(1): 327-350.

[2] Go D E, Stottmann R W. The impact of CRISPR/Cas9-based genomic engineering on biomedical research and medicine. Current molecular medicine, 2016, 16(4): 343-352.

[3] Capecchi M. Altering the genome by homologous recombination. Science, 1989, 244(4910): 1288-1292.

[4] Bellaiche Y, Mogila V, Perrimon N. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics, 1999, 152(3): 1037-1044.

[5] Baker M. Gene-editing nucleases. Nature Methods, 2012, 9(1): 23-26.

[6] Urnov F D, Rebar E J, Holmes M C, Zhang H S, Gregory P D. Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 2010, 11(9): 636-646.

[7] Christian M, Cermak T, Doyle E L, Schmidt C, Zhang F, Hummel A, Bogdanove A J, Voytas D F. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 2010, 186(2): 757-761.

[8] Mali P, Aach J, Stranges P B, Esvelt K M, Moosburner M, Kosuri S, Yang L, Church G M. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology, 2013, 31(9): 833-838.

[9] Zetsche B, Gootenberg J S, Abudayyeh O O, Slaymaker I M, Makarova K S, Essletzbichler P, Volz S E, Joung J, van der Oost J, Regev A, Koonin E V, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015, 163(3): 759-771.

[10] Cohen-Tannoudji M, Robine S, Choulika A, Pinto D, El Marjou F, Babinet C, Louvard D, Jaisser F. I-SceI-induced gene replacement at a natural locus in embryonic stem cells. Molecular and Cellular Biology, 1998, 18(3): 1444-1448.

[11] Symington L S, Gautier J. Double-strand break end resection and repair pathway choice. Annual Review of Genetics, 2011, 45: 247-271.

[12] Bibikova M, Golic M, Golic K G, Carroll D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc- finger nucleases. Genetics, 2002, 161(3): 1169-1175.

[13] Ishii T. Germline genome-editing research and its socioethical implications. Trends in molecular medicine, 2015, 21(8): 473-481.

[14] Weeks D P, Spalding M H, Yang B. Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnology Journal, 2016, 14(2): 483-495.

[15] Yang H, Gao P, Rajashankar K R, Patel D J. PAM- dependent target DNA recognition and cleavage by C2c1 CRISPR- Cas endonuclease. Cell, 2016, 167(7): 1814-1828.

[16] Abudayyeh O O, Gootenberg J S, Konermann S, Joung J, Slaymaker I M, Cox D B T, Shmakov S, Makarova K S, Semenova E, Minakhin L, Severinov K, Regev A, Lander E S, Koonin E V, Zhang F. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 2016, 353(6299): aaf5573.

[17] Zhu C, Bortesi L, Baysal C, Twyman R M, Fischer R, Capell T, Schillberg S, Christou P. Characteristics of genome editing mutations in cereal crops. Trends in Plant Science, 2017, 22(1): 38-52.

[18] 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.

[19] 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.

[20] Shan Q, Zhang Y, Chen K, Zhang K, Gao C. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnology Journal, 2015, 13(6): 791-800.

[21] Ma L, Zhu F, Li Z, Zhang J, Li X, Dong J, Wang T. TALEN-based mutagenesis of lipoxygenase LOX3 enhances the storage tolerance of rice (Oryza sativa) seeds. PLoS One, 2015, 10(12): e0143877.

[22] Blanvillain-Baufume S, Reschke M, Sole M, Auguy F, Doucoure H, Szurek B, Meynard D, Portefaix M, Cunnac S, Guiderdoni E, Boch J, Koebnik R. Targeted promoter editing for rice resistance to Xanthomonas oryzae pv. oryzae reveals differential activities for SWEET14-inducing TAL effectors. Plant Biotechnology Journal, 2017, 15(3): 306-317.

[23] Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, 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.

[24] Haun W, Coffman A, Clasen B M, Demorest Z L, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L, Voytas D F, Zhang F. Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnology Journal, 2014, 12(7): 934-940.

[25] Clasen B M, Stoddard T J, Luo S, Demorest Z L, Li J, Cedrone F, Tibebu R, Davison S, Ray E E, Daulhac A, Coffman A, Yabandith A, Retterath A, Haun W, Baltes N J, Mathis L, Voytas D F, Zhang F. Improving cold storage and processing traits in potato through targeted gene knockout. Plant Biotechnology Journal, 2016, 14: 169-176.

[26] Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y G, Zhao K. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One, 2016, 11(4): e0154027.

[27] 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.

[28] Wang Y, Geng L, Yuan M, Wei J, Jin C, Li M, Yu K, Zhang Y, Jin H, Wang E, Chai Z, Fu X, Li X. Deletion of a target gene in Indica rice via CRISPR/Cas9. Plant Cell Reports, 2017, 36(8): 1333-1343..

[29] Shen L, Wang C, Fu Y, Wang J, Liu Q, Zhang X, Yan C, Qian Q, Wang K. QTL editing confers opposing yield performance in different rice varieties. Journal of integrative plant biology, 2016 Sep 15. doi: 10.1111/jipb.12501.

[30] Xu R, Yang Y, Qin R, Li H, Qiu C, Li L, Wei P, Yang J. Rapid improvement of grain weight via highly efficient CRISPR/Cas9- mediated multiplex genome editing in rice. Journal of Genetics and Genomics, 2016, 43(8): 529-532.

[31] Li Q, Zhang D, Chen M, Liang W, Wei J, Qi Y, Yuan Z. Development of japonica photo-sensitive genic male sterile rice lines by editing carbon starved anther using CRISPR/Cas9. Journal of Genetics and Genomics, 2016, 43(6): 415-419.

[32] Zhou H, He M, Li J, Chen L, Huang Z, Zheng S, Zhu L, Ni E, Jiang D, Zhao B, Zhuang C. Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Scientific Reports, 2016, 6: 37395.

[33] Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y G. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 2015, 8(8): 1274-1284.

[34] Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, Du W, Du J, Francis F, Zhao Y, Xia L. Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in Plant Science, 2017, 8: 298.

[35] Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Molecular Plant, 2016, 9(4): 628-631.

[36] Endo M, Mikami M, Toki S. Biallelic gene targeting in rice. Plant Physiology, 2016, 170(2): 667-677.

[37] 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.

[38] Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nature Biotechnology, 2017, 35(5): 441-443.

[39] Shi J, Gao H, Wang H, Lafitte H R, Archibald R L, Yang M, Hakimi S M, Mo H, Habben J E. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal, 2017, 15(2): 207-216.

[40] Svitashev S, Young J K, Schwartz C, Gao H, Falco S C, Cigan A M. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiology, 2015, 169(2): 931-945.

[41] Svitashev S, Schwartz C, Lenderts B, Young J K, Mark Cigan A. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nature Communications, 2016, 7: 13274.

[42] Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu J L, Gao C. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature Communications, 2016, 7: 12617.

[43] Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soybean. Plant Biotechnology Journal, 2017 May 16. doi: 10.1111/pbi.12758.

[44] Soyk S, Muller N A, Park S J, Schmalenbach I, Jiang K, Hayama R, Zhang L, Van Eck J, Jimenez-Gomez J M, Lippman Z B. Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato. Nature Genetics, 2017, 49(1): 162-168.

[45] Jia H, Zhang Y, Orbovic V, Xu J, White F F, Jones J B, Wang N. Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnology Journal, 2017, 15(7): 817-823..

[46] Waltz E. Gene-edited CRISPR mushroom escapes US regulation. Nature, 2016, 532(7599): 293.

[47] Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J. Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnology Journal, 2017, 15(6): 713-717.

[48] Begemann M B, Gray B N, January E, Gina C Gordon, He Y h, Liu Hvj, Wu X r, Brutnell T P, Mockler T C, Oufattole M. Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases. Scientific Reports, 2017, 7(1): 11606..

[49] Kandavelou K, Chandrasegaran S. Custom-designed molecular scissors for site-specific manipulation of the plant and mammalian genomes. Methods in Molecular Biology, 2009, 544: 617-636.

[50] Kim Y G, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to FokⅠ cleavage domain. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(3): 1156-1160.

[51] Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver R C, Katibah G E, Amora R, Boydston E A, Zeitler B, Meng X, Miller J C, Zhang L, Rebar E J, Gregory P D, Urnov F D, Jaenisch R. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nature Biotechnology, 2009, 27(9): 851-857.

[52] Remy S, Tesson L, Menoret S, Usal C, Scharenberg A M, Anegon I. Zinc-finger nucleases: a powerful tool for genetic engineering of animals. Transgenic Research, 2010, 19(3): 363-371.

[53] Bibikova M, Beumer K, Trautman J K, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science, 2003, 300(5620): 764.

[54] Doyon Y, McCammon J M, Miller J C, Faraji F, Ngo C, Katibah G E, Amora R, Hocking T D, Zhang L, Rebar E J, Gregory P D, Urnov F D, Amacher S L. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnology, 2008, 26(6): 702-708.

[55] Zhang F, Maeder M L, Unger-Wallace E, Hoshaw J P, Reyon D, Christian M, Li X, Pierick C J, Dobbs D, Peterson T, Joung J K, Voytas D F. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(26): 12028-12033.

[56] 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.

[57] Li T, Huang S, Jiang W Z, Wright D, Spalding M H, Weeks D P, Yang B. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Research, 2010, 39(1): 359-372.

[58] Schornack S, Minsavage G V, Stall R E, Jones J B, Lahaye T. Characterization of AvrHah1, a novel AvrBs3-like effector from Xanthomonas gardneri with virulence and avirulence activity. New Phytologist, 2008, 179(2): 546-556.

[59] Boch J, Scholze H, Schornack S, Landgraf A, Hahn S K S, Lahaye T, Nickstadt A, Bonas U. Breaking the code of DNA binding specificity of TAL-type Ⅲ effectors. Science, 2009(326): 1509-1512.

[60] Moscou M J, Bogdanove A J. A simple cipher governs DNA recognition by TAL effectors. Science, 2009, 326(5959): 1501.

[61] Reyon D, Tsai S Q, Khayter C, Foden J A, Sander J D, Joung J K. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnology, 2012, 30(5): 460-465.

[62] Miklis M, Consonni C, Bhat R A, Lipka V, Schulze- Lefert P, Panstruga R. Barley MLO modulates actin-dependent and actin-independent antifungal defense pathways at the cell periphery. Plant Physiology, 2007, 144(2): 1132-1143.

[63] Carter J, Wiedenheft B. SnapShot: CRISPR-RNA-guided adaptive immune systems. Cell, 2015, 163(1): 260-260.

[64] Makarova K S, Koonin E V. Annotation and classification of CRISPR-Cas systems. Methods in Molecular Biology, 2015, 1311: 47-75.

[65] Marraffini L A, Sontheimer E J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature, 2010, 463(7280): 568-571.

[66] Deltcheva E, Chylinski K, Sharma C M, Gonzales K, Chao Y, Pirzada Z A, Eckert M R, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011, 471(7340): 602-607.

[67] Jinek M, Jiang F, Taylor D W, Sternberg S H, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone A T, Charpentier E, Nogales E, Doudna J A. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014, 343(6176): 1247997.

[68] 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.

[69] Hsu P D, Lander E S, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157(6): 1262-1278.

[70] Belhaj K, Chaparro-Garcia A, Kamoun S, Patron N J, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Current Opinion in Biotechnology, 2015, 32: 76-84.

[71] Scheben A, Edwards D. Genome editors take on crops. Science, 2017, 355(6330): 1122-1123.

[72] Liu D f, chen X j, liu j q, ye J c, Guo Z j. The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance. Journal of Experimental Botany, 2012, 63(10): 3899-3912.

[73] Hu Y, Zhang J, Jia H, Sosso D, Li T, Frommer W B, Yang B, White F F, Wang N, Jones J B. Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(4): 521-529.

[74] Okuzaki A, Shimizu T, Kaku K, Kawai K, Toriyama K. A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice. Plant Molecular Biology, 2007, 64(1/2): 219-224.

[75] Shi J, Habben J E, Archibald R L, Drummond B J, Chamberlin M A, Williams R W, Lafitte H R, Weers B P. Overexpression of ARGOS genes modifies plant sensitivity to ethylene, leading to improved drought tolerance in both Arabidopsis and maize. Plant Physiology, 2015, 169(1): 266-282.

[76] Gao P, Yang H, Rajashankar K R, Huang Z, Patel D J. Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Cell Research, 2016, 26(8): 901-913.

[77] Endo A, Masafumi M, Kaya H, Toki S. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Scientific Reports, 2016, 6: 38169.

[78] 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(7600): 517-521.

[79] Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker I M, Li Y, Fedorova I, Nakane T, Makarova K S, Koonin E V, Ishitani R, Zhang F, Nureki O. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell, 2016, 165(4): 949-962.

[80] Dong D, Ren K, Qiu X, Zheng J, Guo M, Guan X, Liu H, Li N, Zhang B, Yang D, Ma C, Wang S, Wu D, Ma Y, Fan S, Wang J, Gao N, Huang Z. The crystal structure of Cpf1 in complex with CRISPR RNA. Nature, 2016, 532(7600): 522-526.

[81] Lei C, Li S Y, Liu J K, Zheng X, Zhao G P, Wang J. The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro. Nucleic Acids Research, 2017, 45(9): e47

[82] Kleinstiver B P, Tsai S Q, Prew M S, Nguyen N T, Welch M M, Lopez J M, McCaw Z R, Aryee M J, Joung J K. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nature Biotechnology, 2016, 34(8): 869-874.

[83] Kim D, Kim J, Hur J K, Been K W, Yoon S h, Kim J S. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nature Biotechnology, 2016, 34(8): 863-868.

[84] Kim H, Kim S T, Ryu J, Kang B C, Kim J S, Kim S G. CRISPR/Cpf1-mediated DNA-free plant genome editing. Nature Communications, 2017, 8: 14406.

[85] Tang X, Lowder L G, Zhang T, Malzahn A A, Zheng X, Voytas D F, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland E R, Zhang Y, Qi Y. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nature Plants, 2017, 3: 17018.

[86] Hu X, Wang C, Liu Q, Fu Y, Wang K. Targeted mutagenesis in rice using CRISPR-Cpf1 system. Journal of Genetics and Genomics, 2017, 44(1): 71-73.

[87] Wang M, Mao Y, Lu Y, Tao X, Zhu J K. Multiplex gene editing in rice using the CRISPR-Cpf1 system. Molecular Plant, 2017, 10(7): 1011-1013..

[88] Komor A C, Kim Y B, Packer M S, Zuris J A, Liu D R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016, 533(7603): 420-424.

[89] Lu Y, Zhu J K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Molecular Plant, 2017, 10(3): 523-525.

[90] Li J, Sun Y, Du J, Zhao Y, Xia L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Molecular Plant, 2017, 10(3): 526-529.

[91] Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu J L, Wang D, Gao C. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nature Biotechnology, 2017, 35(5): 438-440.

[92] Waltz E. CRISPR-edited crops free to enter market, skip regulation. Nature Biotechnology, 2016, 34(6): 582.

[93] 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. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nature Biotechnology, 2015, 33(11): 1162-1164.

[94] Cho S W, Lee J, Carroll D, Kim J S, Lee J. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics, 2013, 195(3): 1177-1180.

[95] Kim S, Kim D, Cho S W, Kim J, Kim J S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.Genome Research, 2014, 24(6): 1012-1019.

[96] Subburaj S, Chung S J, Lee C, Ryu S M, Kim D H, Kim J S, Bae S, Lee G J. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Reports, 2016, 35(7): 1535-1544.

[97] Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications, 2017, 8: 14261.

[98] Cermak T, Doyle E L, Christian M, Wang L, Zhang Y, Schmidt C, Baller J A, Somia N V, Bogdanove A J, Voytas D F. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, 39(12): e82.

[99] Shan Q, Wang Y, Chen K, Liang Z, Li J, Zhang Y, Zhang K, Liu J, Voytas D F, Zheng X, Zhang Y, Gao C. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Molecular Plant, 2013, 6(4): 1365-1368.

[100] Zhang H, Gou F, Zhang J, Liu W, Li Q, Mao Y, Botella J R, Zhu J K. TALEN-mediated targeted mutagenesis produces a large variety of heritable mutations in rice. Plant Biotechnology Journal, 2016, 14: 186-194.

[101] Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu J K. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 2014, 12(6): 797-807.

[102] 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.

[103] Fu Y, Foden J A, Khayter C, Maeder M L, Reyon D, Joung J K, Sander J D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 2013, 31(9): 822-826.

[104] Endo M, Mikami M, Toki S. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant and Cell Physiology, 2015, 56(1): 41-47.

[105] Schaefer K A, Wu W-H, Colgan D F, Tsang S H, Bassuk A G, Mahajan V B. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nature Methods, 2017, 14(6): 547-548.

[106] Khandagale K, Nadaf A. Genome editing for targeted improvement of plants. Plant Biotechnology Reports, 2016, 10(6): 327-343.

[107] Ledford H. US regulation misses some GM crops. Nature, 2013, 500: 389-390.

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

[109] Huang S, Weigel D, Beachy R N, Li J. A proposed regulatory framework for genome-edited crops. Nature Genetics, 2016, 48(2): 109-111.

[110] Where genome editing is needed. Nature Genetics, 2016, 48(2): 103.

[111] Kleinstiver B P, Pattanayak V, Prew M S, Tsai S Q, Nguyen N T, Zheng Z, Joung J K. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 2016, 529(7587): 490-495.

[112] Slaymaker I M, Gao L, Zetsche B, Scott D A, Yan W X, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science, 2016, 351(6268): 84-88.

[113] Gao L, Cox D B T, Yan W X, Manteiga J C, Schneider M W, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F. Engineered Cpf1 variants with altered PAM specificities. Nature Biotechnology, 2017 Jun 05. doi: 10.1038/nbt.3900.

[114] Hu X, Meng X, Liu Q, Li J, Wang K. Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice. Plant Biotechnology Journal, 2017 Jun 12. doi: 10.1111/pbi.12771.

[115] Wang M, Lu Y, Botella J R, Mao Y, Hua K, Zhu J K. Gene targeting by homology-directed repair in rice using a geminivirus- based CRISPR/Cas9 system. Molecular Plant, 2017, 10(7): 1007-1010. .