New Generation Genome Editing Techniques: ZFNs, TALENs, CRISPRs and Their Usage in Plant Research

Yıl 2017, Cilt: 26 Sayı: 1, 111 - 126, 29.06.2017

M. Aydın Akbudak Kübra Kontbay

https://doi.org/10.21566/tarbitderg.323614

Cited By: 4

Öz

ZFNs, TALENs and
CRISPRs are the new generation genome editing tools functioning by binding a
specified target region on DNA. Using one of these techniques, target genes
could be edited by insertion/deletions. It is also possible to replace
nucleotides in target genes via homologous recombination. Compared to the
current methods, new generation genome editing is faster, easier, more
efficient and less expensive. This review covers how and where new generation
genome editing techniques can be used, comparison one to another, and
highlights applications in plant genetic engineering and plant breeding.
Furthermore, the release of genome edited plants to the environment and current
regulations in the world are discussed. It is predicted that improvements in
binding and excising efficacy of new generation genome editing techniques would
take the current research one step further, and would be a major aid to plant
breeding that would lead to generating crop varieties with higher yield,
quality and tolerance to biotic and abiotic stresses.

Anahtar Kelimeler

ZFN, TALEN, CRISPR, gene silencing

Yeni Nesil Genom Düzenleme Teknikleri: ZFN, TALEN, CRISPR’lar ve Bitkilerde Kullanımı

Öz

ZFN, TALEN ve CRISPR’lar
DNA’da hedeflenen bölgeye bağlanabilmeleri sayesinde genomda düzenleme yapmaya imkân
veren yeni nesil genom düzenleme teknikleridir. Bu tekniklerin kullanımı sayesinde
hedef genler mutasyona uğratılarak ya da genomdan kesilerek susturulabilmekte, ayrıca
genlerde istenilen nükleotidlerin değiştirilmesi de mümkün olabilmektedir. Yapılan
genom düzenlemeleri hâlihazırda kullanılan metotlara kıyasla daha hızlı, daha kolay,
etkin ve ucuzdur. Bu makalede yeni nesil genom düzenleme tekniklerinin çalışma
prensipleri, kullanıldıkları alanlar, tekniklerin birbirleriyle kıyaslanması ve
bu teknikler kullanılarak genetik mühendisliği ve ıslahı alanında şu ana kadar yapılmış
çalışmalara yer verilmiştir. Makalenin son bölümünde bu tekniklerle yapılan
düzenlemeler sonucunda elde edilen bitkilerin doğaya salınımı ve bu konuda mevcut
düzenlemelere de değinilmiştir. Yeni nesil genom düzenleme tekniklerinin bağlanma
ve kesim etkinliklerinin artırılması mevcut çalışmaları bir adım daha ileriye taşıyarak,
daha verimli, kaliteli ve çevresel faktörlere karşı toleranslı çeşitlerin
geliştirilmesine imkân tanıyacaktır.

Anahtar Kelimeler

ZFN, TALEN, CRISPR, gen susturma

Kaynakça

• Alagoz Y., Gurkok T., Zhang BH. and Unver T., 2016. Manipulating the Biosynthesis of Bioactive Compound Alkaloids for Next-Generation Metabolic Engineering in Opium Poppy Using CRISPR-Cas 9 Genome Editing Technology, Scientific Reports 6, Article number: 30910 (DOI:10.1038/srep30910)
• Araki M. and Ishii T., 2015. Towards social acceptance of plant breeding by genome editing. Trends in Plant Science, 20(3):145-149
• Baltes N.J. and Voytas DF., 2015. Enabling plant synthetic biology through genome engineering. Trends in Biotechnology, 33(2):120-131
• Belhaj K., Chaparro-Garcia A., Kamoun S., Patron NJ. and Nekrasov V., 2015. Editing plant genomes with CRISPR/Cas9. Current Opinion in Biotechnology, 32:76-84
• Boch J., Scholze H., Schornack S., Landgraf A., Hahn S., Kay S., Lahaye T., Nickstadt A. and Bonas U., 2009. Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science, 326(5959):1509-1912 Bortesi L. and Fischer R., 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 33(1):41-52
• Budhagatapalli N., Rutten T., Gurushidze M., Kumlehn J. and Hensel G., 2015. Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley. G3-Genes Genomes Genetics, 5(9):1857-1863
• Butler N.M., Atkins PA., Voytas D.F. and Douches D.S., 2015. Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System. Plos One 10(12): e0144591. https://doi.org/10.1371/journal.pone.0144591
• Carroll D., 2011. Efficient Genome Engineering with Zinc-finger Nucleases. Invitro Cellular & Developmental Biology-Animal, 188(4):773-782
• Ceasar S.A., Rajan V., Prykhozhij S.V., Berman J.N. and Ignacimuthu S., 2016. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9. Biochimica Et Biophysica Acta-Molecular Cell Research, 1863(9):2333-2344
• Cermak T., Baltes N.J., Cegan R., Zhang Y. and Voytas D.F., 2015. High-frequency, precise modification of the tomato genome, Genome Biology 16:232 (DOI:10.1186/s13059-015-0796-9)
• Cermak T., Doyle E.L., Christian M., Wang L., Zhang Y., Schmidt C., Baller J.A., Somia N.V., Bogdanve A.J. and Voytas D.F., 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 39(17):7879 (DOI:10.1093/nar/gkr739)
• Chandrasekaran J., Brumin M., Wolf D., Leibman D., Klap C., Pearlsman M., Sherman A., Arazi T. and Gal-On A., 2016. Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology. Molecular plant pathology, 17.7 (2016): 1140-1153
• Char S.N., Unger-Wallace E., Frame B., Briggs S.A., Main M., Spalding M.H., Vollbrecht E., Wang K. and Yang B., 2015. Heritable site-specific mutagenesis using TALENs in maize. Plant Biotechnology Journal, 13(7):1002-1010
• 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. and Zhang F., 2016. Improving cold storage and processing traits in potato through targeted gene knockout. Plant Biotechnology Journal, 14(1):169-176
• Collonnier C., Epert A., Mara K., Maclot F., Maclot F., Guyon-Debast A., Charlot F., Charles White C., Schaefer D.G., and Nogué F., 2016. CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens. Plant Biotechnology Journal 15(1):122-131. (DOI: 10.1111/pbi.12596)
• Cong L., Ran FA., Cox D., Lin S.L., Barretto R., Habib N., Hsu P.D., Wu X., Jiang W., Marraffini L.A. and Zhang F., 2013. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science, 339(6121):819-823 (DOI: 10.1126/science.1231143)
• De Pater S., Neuteboom L.W., Pinas J.E., Hooykaas P.J.J and van der Zaal B.J., 2009. ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnology Journal, 7(8):821-835 (DOI: 10.1111/j.1467-7652.2009.00446.x.)
• Ding Y.D., Li H., Chen L.L. and Xie K.B., 2016. Recent Advances in Genome Editing Using CRISPR/Cas9. Frontiers in Plant Science, 7:703
• Dionisio G., Madsen C.K., Holm P.B., Welinder K.G., Jørgensen M., Stoger E., Arcalis E. and Brinch-Pedersen H., 2011. Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize, and Rice. Plant Physiology, 156(3):1087-100 (DOI: 10.1104/pp.110.164756)
• Endo M., Mikami M. and Toki S., 2015. Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice. Plant and Cell Physiology, 56(1):41-47
• Fan D., Liu T.T., Li C.F., Jiao B., Li S., Hou Y.S. and Luoa K., 2015. Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation. Scientific Reports, 5:12217 (DOI: 10.1038/srep12217)
• Fichtner F., Castellanos R.U. and Ulker B., 2014. Precision genetic modifications: a new era in molecular biology and crop improvement. Planta, 239(4):921-39
• Gaj T., Gersbach C.A. and Barbas C.F., 2013. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology, 31(7):397-405
• Gasiunas G., Barrangou R., Horvath P. and Siksnys V., 2012. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109(39):E2579-E86
• Hartung F. and Schiemann J., 2014. Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU. Plant Journal, 78(5):742-52
• Haun W., Coffman A., Clasen BM., Demorest Z.L., Lowy A., Ray E., Retterath A., Stoddard T., Juillerat A., Cedrone F., Mathis L., Voytas D.F. and Zhang F., 2014 Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnology Journal, 12(7):934-940 (DOI: 10.1111/pbi.12201)
• Heap B., 2013 Europe should rethink its stance on GM crops. Nature. 498(7455):409 (DOİ:10.1038/498409a.)
• Huang S., Weigel D., Beachy RN. and Li J., 2016. A proposed regulatory framework for genome-edited crops. Nature Genetics, 48(2):109-111
• Hwang W.Y., Fu Y.F., Reyon D., Maeder M.L., Tsai S.Q., Sander J.D., Peterson R.T., Yeh J-R J., and Joung J.K., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology, 31(3):227-229 (DOI:10.1038/nbt.2501)
• Ito Y., Nishizawa-Yokoi A., Endo M., Mikami M. and Toki S. 2015. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochemical and Biophysical Research Communications, 467(1):76-82
• Jia H.G. and Wang N., 2014. Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA. Plos One, 9(4): e93806 (DOI:10.1371/journal.pone.0093806)
• Jia H., Zhang Y., Orbović V., Xu J., White F.F., Jones J.B. and Wang N., 2016. Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnology Journal, DOI: 10.1111/pbi.12677
• Jiang W.Z., Brueggeman A.J., Horken K.M., Plucinak T.M. and Weeks D.P., 2014. Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii. Eukaryotic Cell, 13(11):1465-1469
• Jiang W.Z., Zhou H.B., Bi H.H., Fromm M., Yang B. and Weeks D.P. 2013. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research, 41(20): e188. (DOI: 0.1093/nar/gkt780)
• Jiang W.Z., Henry I.M., Lynagh P.G., Comai L., Cahoon E.B. and Weeks D.P., 2016. Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnology Journal, 15: 648–657. (DOI:10.1111/pbi.12663)
• Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A. and Charpentier E., 2012. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096):816-821
• Joung J.K. and Sander J.D., 2013. INNOVATION TALENs: a widely applicable technology for targeted genome editing. Nature Reviews Molecular Cell Biology, 14(1):49-55
• Kumar V. and Jain M., 2015. The CRISPR-Cas system for plant genome editing: advances and opportunities. Journal of Experimental Botany, 66(1):47-57
• Lawrenson T., Shorinola O., Stacey N., Li C.D., Ostergaard L., Patron N., Uauy C. and Harwood W., 2015. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biology, 16(1):258 (DOI 10.1186/s13059-015-0826-7)
• Lee H.B., Sundberg B.N., Sigafoos A.N. and Clark K.J., 2016. Genome Engineering with TALE and CRISPR Systems in Neuroscience. Frontiers in Genetics, 7 (DOI: 10.3389/fgene.2016.00047)
• Li T., Liu B., Spalding M.H., Weeks D.P. and Yang B., 2012. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nature Biotechnology, 30(5):390-392
• Lopez-Obando M., Hoffmann B., Géry C., Guyon Debast A., Téoulé E, Rameau C, Bonhomme S, and Nogué F., 2016. Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens. G3: Genes Genomes Genetics, 6(11): 3647-3653 (DOİ: 10.1534/g3.116.033266)
• Lor V.S., Starker C.G., Voytas D.F., Weiss D. and Olszewski N.E., 2014. Targeted Mutagenesis of the Tomato PROCERA Gene Using Transcription Activator-Like Effector Nucleases. Plant Physiology, 166(3):1288-1291
• Lowder L.G., Zhang D.W., Baltes N.J., Paul J.W., Tang X., Zheng X.L., Voytas D.F., Hsieh T.-F., Zhang Y. and Qi Y., 2015. A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiology, 169(2):971-985 (DOI: 10.1104/pp.15.00636)
• Ma X.L., Zhu QL., Chen Y.L. and Liu Y.G., 2016. CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications. Molecular Plant 9(7):961-74
• Mahfouz M.M., Piatek A. and Stewart C.N., 2014. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives. Plant Biotechnology Journal, 12(8):1006-1014
• Malnoy M., Viola R., Jung M.H., Koo O.J., Kim S., Kim J.S., Velasco R. and Kanchiswamy C.N., 2016. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 Ribonucleoproteins. Frontiers in Plant Science, 7:1904 (DOI=10.3389/fpls.2016.01904)
• Mao YF., Zhang H., Xu NF., Zhang BT., Gou F. and Zhu JK., 2013. Application of the CRISPRCas System for Efficient Genome Engineering in Plants. Molecular Plant, 6(6):2008-2011
• Michno J.M., Wang X.B., Liu J.Q., Curtin S.J., Kono T.J.Y. and Stupar R.M., 2015. CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops & Food-Biotechnology in Agriculture and the Food Chain, 6(4):243-52
• Miller J.C., Holmes M.C., Wang J.B., Guschin D.Y., Lee Y.L., Rupniewski I., Beausejour C.M., Waite A.J., Wang N.S., Kim K.A., Philip D., Gregory P.D., Pabo C.O, and Rebar E.J., 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nature Biotechnology 25(7):778-85 (DOI:10.1038/nbt1319)
• Miller J.C., Tan S., Qiao G., Barlow K.A. and Wang J., 2011. A TALE nuclease architecture for efficient genome editing. Nature biotechnology, 1;29(2):143-148.
• Moscou M.J. and Bogdanove A.J., 2009. A Simple Cipher Governs DNA Recognition by TAL Effectors. Science, 326(5959):1501 Nejat N., Rookes J., Mantri N.L. and Cahill D.M., 2016. Plant–pathogen interactions: toward development of next-generation disease-resistant plants. Critical reviews in biotechnology, 37(2):229-237. (DOI: 10.3109/07388551)
• Nekrasov V., Staskawicz B., Weigel D., Jones J.D.G. and Kamoun S., 2013. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature Biotechnology, 31(8):691-693
• Nishitani C., Hirai N., Komori S., Wada M., Okada K., Osakabe K. Yamamoto T. and Osakabea Y., 2016. Efficient Genome Editing in Apple Using a CRISPR/Cas9 system. Scientific Reports 6, Article number: 31481 (DOI: 10.1038/srep31481)
• Osakabe Y. and Osakabe K., 2015. Genome Editing with Engineered Nucleases in Plants. Plant and Cell Physiology, 56(3):389-400
• Pan C., Ye L., Qin L., Liu X., He Y.J., Wang J., Chen L. and Lua G., 2016. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports 6:24765 (DOI: 10.1038/srep24765)
• Pattanayak V., Guilinger J.P. and Liu D.R., 2014. Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes. Use of CRISPR/Cas9, ZFNS, and TALENs in Generating Site-Specific Genome Alterations. Methods in Enzymology, 546:47-78
• Paul J.W. and Qi Y.P., 2016. CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects. Plant Cell Reports, 35(7):1417-1427
• Petolino J.F., 2015 Genome editing in plants via designed zinc finger nucleases. Invitro Cellular and Developmental Biology-Plant, 51(1):1-8 (DOI: 10.1016/j.cell.2013.02.022).
• Qi L.S., Larson M.H., Gilbert L.A., Doudna J.A., Weissman J.S., Arkin A.P. and Lim W.A., 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152(5):1173-1183 (DOI: 10.1016/j.cell.2013.02.022).
• Ramalingam S., Kandavelou K., Rajenderan R. and Chandrasegaran S. 2011. Creating Designed Zinc-Finger Nucleases with Minimal Cytotoxicity. Journal of Molecular Biology, 405(3):630-41
• Rani R., Yadav P., Barbadikar K.M., Baliyan N., Malhotra E.V., Singh B.K., Kumar A. and Singh D., 2016. CRISPR/Cas9: a promising way to exploit genetic variation in plants. Biotechnology Letters, 38(12):1991-2006 (DOI: 10.1007/s10529-016-2195-z)
• Ray A. and Langer M., 2002. Homologous recombination: ends as the means. Trends in Plant Science, 7(10):435-40 Ren C., Liu XJ., Zhang Z., Wang Y., Duan W., Li S.H. and Liang Z., 2016. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Scientific Reports 6:32289 (DOI: 10.1038/srep32289).
• Reyon D., Tsai S.Q., Khayter C., Foden J.A., Sander J.D. and Joung J.K., 2012. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnology, 30(5):460-465
• Samanta MK., Dey A. and Gayen S., 2016. CRISPR/Cas9: an advanced tool for editing plant genomes. Transgenic Research, 25(5):561-73
• Sander J.D., Dahlborg E.J., Goodwin M.J., Cade L., Zhang F., Cifuentes D., Curtin S.J., Blackburn J.S., Thibodeau-Beganny S., Qi Y., Pierick C.J., Hoffman E., Maeder M.L., Khayter C., Reyon D., Dobbs D., Langenau D.M., Stupar R.M., Giraldez A.J., Voytas D.F., Peterson R.T., Yeh J.R. and Joung J.K., 2011. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nature Methods, 8(1):67-69 (DOI: 10.1038/nmeth.1542)
• Schaeffer S.M. and Nakata P.A., 2015. CRISPR/Cas9-mediated genome editing and gene replacement in plants: Transitioning from lab to field. Plant Science, 240:130-42
• Schaeffer S.M. and Nakata P.A., 2016. The expanding footprint of CRISPR/Cas9 in the plant sciences. Plant Cell Reports, 35(7):1451-68
• Schiml S., Fauser F. and Puchta H., 2014. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant Journal, 80(6):1139-1150 (DOI: 10.1111/tpj.12704)
• Schiml S. and Puchta H., .2016. Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. Plant Methods, 12:8 (DOI: 10.1186/s13007-016-0103-0)
• Schneider K., Schiermeyer A., Dolls A., Koch N., Herwartz D., Kirchhoff J., Rainer F., Sean M. R., Zehui C., David R.C., Lakshmi S.D., Michael A.W., Steven W.R., Helga S. and Stefan S., 2016. Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut. Plant Biotechnology Journal, 14(4):1151-1160 (DOI: 10.1111/pbi.12483) Scholze H. and Boch J., 2011. TAL effectors are remote controls for gene activation. Current Opinion in Microbiology, 14(1):47-53
• Shan Q., Wang Y., Chen K., Liang Z., Li J., Zhang Y., Zhang K., Liu J., Voytas D.F., Zheng X., Zhang Y. and Gao C., 2013. Rapid and Efficient Gene Modification in Rice and Brachypodium Using TALENs. Molecular Plant, 6(4):1365-1368
• Shan QW., Zhang Y., Chen KL., Zhang K. and Gao CX., 2015. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnology Journal, 13(6):791-800(DOI: 10.1111/pbi.12312)
• Shen H., Strunks GD., Klemann B.J., Hooykaas P.J. and de Pater S., 2016. CRISPR/Cas9-Induced Double Strand Break Repair in Arabidopsis Non-homologous End-Joining Mutants. G3: Genes Genomes Genetics, 7(1):193-202
• Shin S.E., Lim J.M., Koh H.G., Kim E.K., Kang N.K., Jeon S., Kwon S., Shin W.S., Lee B., Hwangbo K., Kim J., Ye S.H., Yun J.Y., Seo H., Oh H.M., Kim K.J., Kim J.S., Jeong W.J., Chang Y.K., and Jeong B.R., 2016. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Scientific Reports 6:27810. (DOI: 10.1038/srep27810)
• 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. and Urnov F.D., 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459(7245):437-441 (DOI: 10.1038/nature07992).
• Sonoda E., Hochegger H., Saberi A., Taniguchi Y. and Takeda S., 2006. Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair, 5(9-10):1021-1029
• Sugano S.S., Shirakawa M., Takagi J., Matsuda Y., Shimada T., Hara-Nishimura I. and Kohchi T., 2014. CRISPR/Cas9-Mediated Targeted Mutagenesis in the Liverwort Marchantia polymorpha L. Plant and Cell Physiology, 55(3):475-481
• Sun Y.W., Zhang X., Wu CY., He YB., Ma Y., Hou H., Guo X., Du W., Zhao Y. and Xia L., 2016. Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase. Molecular Plant, 9(4):628-631 (DOI: 10.1016/j.molp.2016.01.001)
• Svitashev S., Schwartz C., Lenderts B., Young J.K. and Cigan A.M., 2016. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nature Communications, 7, Article number: 13274 (DOI: 10.1038/ncomms13274)
• Szczepek M., Brondani V., Buchel J., Serrano L., Segal D.J. and Cathomen T., 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nature Biotechnology, 25(7):786-93
• Takata M., Sasaki M.S., Sonoda E., Morrison C., Hashimoto M., Utsumi H., Yamaguchi-Iwai Y., Shinohara A. and Takeda S., 1998. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. Embo. Journal, 17(18):5497-5508 (DOI: 10.1093/emboj/17.18.5497)
• Townsend J.A., Wright D.A., Winfrey R.J., Fu F.L., Maeder M.L., Joung J.K. and Voytas D.F., 2009. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature, 459(7245):442-445 (DOI: 10.1038/nature07845)
• Urnov F.D., Rebar E.J., Holmes M.C., Zhang H.S. and Gregory P.D., 2010. Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9):636-46
• Voytas D.F., 2013. Plant Genome Engineering with Sequence-Specific Nucleases. Annual Review of Plant Biology, 64: 327-50
• Voytas D.F. and Gao C.X., 2014. Precision Genome Engineering and Agriculture: Opportunities and Regulatory Challenges. Plos Biology, 12(6): e100187 (DOI: 10.1371/journal.pbio.1001877)
• Wah D.A., Bitinaite J., Schildkraut I. and Aggarwal A.K., 1998. Structure of FokI has implications for DNA cleavage. Proceedings of the National Academy of Sciences of the United States of America, 95(18):10564-10569
• Wang C., Shen L., Fu Y.P., Yan C.J. and Wang K.J., 2015a. A Simple CRISPR/Cas9 System for Multiplex Genome Editing in Rice. Journal of Genetics and Genomics, 42(12):703-706
• Wang S.H., Zhang S.B., Wang W.X., Xiong X.Y., Meng F.R. and Cui X., 2015b. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Reports, 34(9):1473-1476
• Wang Y.P., Cheng X., Shan Q.W., Zhang Y., Liu J.X., Gao C.X., and Qiu J.L., 2014. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32(9):947-951
• Wang Y., Liu X.J., Ren C., Zhong G.Y., Yang L., Li S.H. and Liang Z., 2016. Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome. BMC Plant Biology, 16:96 (DOI: 10.1186/s12870-016-0787-3)
• Weeks D.P., Spalding M.H. and Yang B., 2016. Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnology Journal 14(2):483-95
• Wendt T., Holm P.B., Starker C.G., Christian M., Voytas D.F., Brinch-Pedersen H. and Holme I.B., 2013. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants. Plant Molecular Biology 83(3):279-285 (DOI: 10.1007/s11103-013-0078-4)
• Wolt J.D., Wang K. and Yang B., 2016. The Regulatory Status of Genome-edited Crops. Plant Biotechnology Journal 14(2):510-518
• Woo J.W., Kim J., Kwon S., Corvalán C., Cho S.W., Kim H., Kim S.G., Kim S.T., Choe S. and Kim J.S., 2015. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nature Biotechnology, 33(11):1162-1164. (DOI: 10.1038/nbt.3389)
• Wu J., Kandavelou K. and Chandrasegaran S., 2007. Custom-designed zinc finger nucleases: What is next? Cellular and Molecular Life Sciences, 64(22):2933-2944
• Xing H.L., Dong L., Wang Z.P., Zhang H.Y., Han C.Y., Liu B, Wang X.C. and Chen QJ., 2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology, 14.1 (2014): 327(DOI: 10.1186/s12870-014-0327-y)
• Zhang B., Yang X., Yang C.P, Li M.Y. and Guo Y.L., 2016a. Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia. Scientific Reports 6 Article number: 20315 (DIO: doi:10.1038/srep20315)
• Zhang D.D., Li Z.X. and Li J.F., 2016b. Targeted Gene Manipulation in Plants Using the CRISPR/Cas Technology. Journal of Genetics and Genomics, 43(5):251-62
• Zhang H., Zhang J.S., Wei P.L., Zhang B.T., Gou F., Feng Z.Y., Mao Y., Yang L., Zhang H., Xu N. and Zhu J.K., 2014. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 12(6):797-807 (DOI: 10.1111/pbi.12200)
• Zhang Y., Liang Z., Zong Y., Wang YP., Liu JX., Chen K.L., Chen, Qiu J.L. and Gao C., 2016c. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature Communications, 7 Article number: 12617 (DOI: 10.1038/ncomms12617)
• Zhang Z.J., Mao Y.F., Ha S., Liu W.S., Botella J.R. and Zhu J.K., 2016d. A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Reports 35(7):1519-1533
• Zhou H.B., Liu B., Weeks D.P., Spalding M.H. and Yang B., 2014. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Research 42(17):10903-10914