Kurawa Ibrahim


Crop improvement as an innovation in plant breeding and genetics requires the deployment of new allelic variants. To achieve this, different types of genome modifications are recently in used, such as ZFN, TALEN, MN, and CRISPR/Cas genome editing systems. However, off-target mutations are the major concerned associated with the use of ZFNs for genome editing. As such, the creation of obligate heterodimeric ZFN architectures that rely on a charge-charge repulsion to prevent unwanted homodimerization of the FokI cleavage domain has been in used to enhance ZFNs specificity. TALENs offer distinct advantages for genome editing compared to ZFNs; it has higher specificity and reduced toxicity compared to some ZFNs and no selection or directed evolution is necessary to engineer TALE arrays. Compared with ZFNs and TALENs, the CRISPR/Cas system is characterized by its simplicity, efficiency, and low cost, and by its ability to target multiple genes. Due to these characteristic features, CRISPR/Cas9 has been rapidly exploited in plants and may be an effective solution to a variety of problems in plant breeding. Conclusively, the CRISPR/Cas9 system provides a valuable platform for generating mutants with high frequency in polyploid crops and very useful for post-transcriptional control of gene expression as well as the simultaneous editing of multiple target sites.

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Ainley, W. M., Sastry‐Dent, L., Welter, M. E., Murray, M. G., Zeitler, B., Amora, R., . . . Strange, T. L. (2013). Trait stacking via targeted genome editing. Plant Biotechnology Journal, 11(9): 1126-1134.

Ali, Z., Abulfaraj, A., Idris, A., Ali, S., Tashkandi, M., & Mahfouz, M. M. (2015). CRISPR/Cas9-mediated viral interference in plants. Genome Biology, 16(1), 238.

Anders, C., Niewoehner, O., Duerst, A., & Jinek, M. (2014). Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513(7519): 569.

Andolfo, G., Iovieno, P., Frusciante, L., & Ercolano, M. R. (2016). Genome-editing technologies for enhancing plant disease resistance. Frontiers in plant science, 7, 1813.

Bibikova, M., Carroll, D., Segal, D. J., Trautman, J. K., Smith, J., Kim, Y.-G., & Chandrasegaran, S. (2001). Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Molecular and cellular biology, 21(1): 289-297.

Boch, J., & Bonas, U. (2010). Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annual Review of Phytopathology, 48: 419-436.

Boissel, S., Jarjour, J., Astrakhan, A., Adey, A., Gouble, A., Duchateau, P., . . . Baker, D. (2013). megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic acids research, 42(4): 2591-2601.

Bortesi, L., & Fischer, R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 33(1): 41-52.

Briggs, A. W., Rios, X., Chari, R., Yang, L., Zhang, F., Mali, P., & Church, G. M. (2012). Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic acids research, 40(15): e117-e117.

Carroll, D. (2011). Genome engineering with zinc-finger nucleases. Genetics, 188(4), 773-782.

Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., . . . Voytas, D. F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research, 39(12): e82-e82.

Char, S. N., Unger‐Wallace, E., Frame, B., Briggs, S. A., Main, M., Spalding, M. H., . . . Yang, B. (2015). Heritable site‐specific mutagenesis using TALEN s in maize. Plant Biotechnology Journal, 13(7): 1002-1010.

Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., & Kim, J.-S. (2014). Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Research, 24(1): 132-141.

Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., . . . Marraffini, L. A. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121): 819-823.

Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., & Liu, D. R. (2015). Small molecule–triggered Cas9 protein with improved genome-editing specificity. Nature chemical biology, 11(5): 316.

Doyon, Y., Vo, T. D., Mendel, M. C., Greenberg, S. G., Wang, J., Xia, D. F., . . . Holmes, M. C. (2011). Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nature methods, 8(1): 74.

Durai, S., Bosley, A., Abulencia, A. B., Chandrasegaran, S., & Ostermeier, M. (2006). A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions. Combinatorial chemistry & high throughput screening, 9(4): 301-311.

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

Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology, 32(3): 279.

Gaj, T., Guo, J., Kato, Y., Sirk, S. J., & Barbas III, C. F. (2012). Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nature methods, 9(8): 805.

Gaj, T., Liu, J., Anderson, K. E., Sirk, S. J., & Barbas III, C. F. (2014). Protein Delivery Using Cys2–His2 Zinc-Finger Domains. ACS chemical biology, 9(8): 1662-1667.

Gao, W., Long, L., Tian, X., Xu, F., Liu, J., Singh, P. K., . . . Song, C. (2017). Genome editing in cotton with the CRISPR/Cas9 system. Frontiers in plant science, 8: 1364.

Guo, J., Gaj, T., & Barbas III, C. F. (2010). Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. Journal of molecular biology, 400(1): 96-107.

Gupta, A., Christensen, R. G., Rayla, A. L., Lakshmanan, A., Stormo, G. D., & Wolfe, S. A. (2012). An optimized two-finger archive for ZFN-mediated gene targeting. Nature methods, 9(6): 588.

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

Heigwer, F., Kerr, G., & Boutros, M. (2014). E-CRISP: fast CRISPR target site identification. Nature methods, 11(2): 122.

Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., . . . Shalem, O. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 31(9): 827.

Hubbard, B. P., Badran, A. H., Zuris, J. A., Guilinger, J. P., Davis, K. M., Chen, L., . . . Liu, D. R. (2015). Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nature methods, 12(10): 939.

Ji, X., Zhang, H., Zhang, Y., Wang, Y., & Gao, C. (2015). Establishing a CRISPR–Cas-like immune system conferring DNA virus resistance in plants. Nature Plants, 1(10): 15144.

Jiang, F., Taylor, D. W., Chen, J. S., Kornfeld, J. E., Zhou, K., Thompson, A. J., . . . Doudna, J. A. (2016). Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science, 351(6275): 867-871.

Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3): 233.

Jiang, W. Z., Henry, I. M., Lynagh, P. G., Comai, L., Cahoon, E. B., & Weeks, D. P. (2017). Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnology Journal, 15(5): 648-657.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096): 816-821.

Joung, J. K., & Sander, J. D. (2013). TALENs: a widely applicable technology for targeted genome editing. Nature reviews Molecular cell biology, 14(1): 49.

Kannan, B., Jung, J. H., Moxley, G. W., Lee, S. M., & Altpeter, F. (2018). TALEN‐mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield. Plant Biotechnology Journal, 16(4): 856-866.

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

Kim, Y., Kweon, J., Kim, A., Chon, J. K., Yoo, J. Y., Kim, H. J., . . . Chung, E. (2013). A library of TAL effector nucleases spanning the human genome. Nature Biotechnology, 31(3): 251.

Kumar, V., & Jain, M. (2014). The CRISPR–Cas system for plant genome editing: advances and opportunities. Journal of experimental botany, 66(1): 47-57.

Li, C., Hao, M., Wang, W., Wang, H., Chen, F., Chu, W., . . . Hu, Q. (2018). An efficient CRISPR/Cas9 platform for rapidly generating simultaneous mutagenesis of multiple gene homoeologs in allotetraploid oilseed rape. Frontiers in plant science, 9.

Li, M., Li, X., Zhou, Z., Wu, P., Fang, M., Pan, X., . . . Li, H. (2016). Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in plant science, 7:377.

Li, T., Liu, B., Spalding, M. H., Weeks, D. P., & Yang, B. (2012). High-efficiency TALEN-based gene editing produces disease-resistant rice. Nature Biotechnology, 30(5): 390.

Lin, J., Chen, H., Luo, L., Lai, Y., Xie, W., & Kee, K. (2014). Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells. Nucleic acids research, 43(2): 1112-1122.

Lin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., . . . Bao, G. (2014). CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic acids research, 42(11): 7473-7485.

Liu, J., Gaj, T., Patterson, J. T., Sirk, S. J., & Barbas III, C. F. (2014). Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PloS one, 9(1): e85755.

Liu, J., Gaj, T., Wallen, M. C., & Barbas III, C. F. (2015). Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering. Molecular Therapy-Nucleic Acids, 4: e232.

Liu, J., Gaj, T., Yang, Y., Wang, N., Shui, S., Kim, S., . . . Barbas III, C. F. (2015). Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nature protocols, 10(11): 1842.

Liu, Y., Ma, S., Wang, X., Chang, J., Gao, J., Shi, R., . . . Zhao, P. (2014). Highly efficient multiplex targeted mutagenesis and genomic structural variation in Bombyx mori cells using CRISPR/Cas9. Insect biochemistry and molecular biology, 49: 35-42.

Lowder, L., Malzahn, A., & Qi, Y. (2016). The rapid evolution of manifold CRISPR systems for plant genome editing. Frontiers in plant science, 7: 1683.

MacDonald, I. C., & Deans, T. L. (2016). Tools and applications in synthetic biology. Advanced drug delivery reviews, 105: 20-34.

Maggio, I., Stefanucci, L., Janssen, J. M., Liu, J., Chen, X., Mouly, V., & Gonçalves, M. A. (2016). Selection-free gene repair after adenoviral vector transduction of designer nucleases: the rescue of dystrophin synthesis in DMD muscle cell populations. Nucleic acids research, 44(3): 1449-1470.

Mak, A. N.-S., Bradley, P., Cernadas, R. A., Bogdanove, A. J., & Stoddard, B. L. (2012). The crystal structure of TAL effector PthXo1 bound to its DNA target. Science, 335(6069): 716-719.

Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., . . . Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science, 339(6121): 823-826.

Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F., & Zhu, J.-K. (2013). Application of the CRISPR–Cas system for efficient genome engineering in plants. Molecular Plant, 6(6): 2008-2011.

Martínez-Fortún, J., Phillips, D. W., & Jones, H. D. (2017). The potential impact of genome editing in world agriculture. Emerging Topics in Life Sciences, 1(2): 117-133.

Meckler, J. F., Bhakta, M. S., Kim, M.-S., Ovadia, R., Habrian, C. H., Zykovich, A., . . . Elsäesser, J. (2013). Quantitative analysis of TALE–DNA interactions suggests polarity effects. Nucleic acids research, 41(7): 4118-4128.

Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., . . . Qu, L.-J. (2013). Targeted mutagenesis in rice using a CRISPR-Cas system. cell research, 23(10): 1233.

Miller, J. C., Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., . . . Hinkley, S. J. (2011). A TALE nuclease architecture for efficient genome editing. Nature Biotechnology, 29(2): 143.

Mock, U., Machowicz, R., Hauber, I., Horn, S., Abramowski, P., Berdien, B., . . . Fehse, B. (2015). mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic acids research, 43(11): 5560-5571.

Mohanta, T. K., Bashir, T., Hashem, A., & Abd_Allah, E. F. (2017). Systems biology approach in plant abiotic stresses. Plant physiology and biochemistry, 121: 58-73.

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

Mussolino, C., Alzubi, J., Fine, E. J., Morbitzer, R., Cradick, T. J., Lahaye, T., Cathomen, T. (2014). TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic acids research, 42(10): 6762-6773.

Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., . . . Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 156(5): 935-949.

Osakabe, K., Osakabe, Y., & Toki, S. (2010). Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases. Proceedings of the National Academy of Sciences, 107(26): 12034-12039.

Osborn, M. J., Webber, B. R., Knipping, F., Lonetree, C.-l., Tennis, N., DeFeo, A. P., . . . Merkel, S. (2016). Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases. Molecular Therapy, 24(3): 570-581.

Pattanayak, V., Ramirez, C. L., Joung, J. K., & Liu, D. R. (2011). Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nature methods, 8(9): 765.

Pavletich, N. P., & Pabo, C. O. (1991). Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science, 252(5007): 809-817.

Rahdar, M., McMahon, M. A., Prakash, T. P., Swayze, E. E., Bennett, C. F., & Cleveland, D. W. (2015). Synthetic CRISPR RNA-Cas9–guided genome editing in human cells. Proceedings of the National Academy of Sciences, 112(51): E7110-E7117.

Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., . . . Zhang, Y. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154(6): 1380-1389.

Ran, Y., Liang, Z., & Gao, C. (2017). Current and future editing reagent delivery systems for plant genome editing. Science China Life Sciences, 60(5): 490-505.

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

Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4): 347.

Sather, B. D., Ibarra, G. S. R., Sommer, K., Curinga, G., Hale, M., Khan, I. F., . . . Sahni, J. (2015). Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Science translational medicine, 7(307): 307ra156-307ra156.

Schmid-Burgk, J. L., Schmidt, T., Kaiser, V., Höning, K., & Hornung, V. (2013). A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nature Biotechnology, 31(1): 76.

Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., . . . Doudna, J. A. (2015). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proceedings of the National Academy of Sciences, 112(33): 10437-10442.

Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., . . . Qiu, J.-L. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology, 31(8): 686.

Shukla, V. K., Doyon, Y., Miller, J. C., DeKelver, R. C., Moehle, E. A., Worden, S. E., . . . Meng, X. (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459(7245): 437.

Smith, A. M., Takeuchi, R., Pellenz, S., Davis, L., Maizels, N., Monnat, R. J., & Stoddard, B. L. (2009). Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proceedings of the National Academy of Sciences, 106(13): 5099-5104.

Smith, J., Bibikova, M., Whitby, F. G., Reddy, A., Chandrasegaran, S., & Carroll, D. (2000). Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic acids research, 28(17): 3361-3369.

Sorek, R., Lawrence, C. M., & Wiedenheft, B. (2013). CRISPR-mediated adaptive immune systems in bacteria and archaea. Annual review of biochemistry, 82: 237-266.

Stoddard, B. L. (2011). Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure, 19(1): 7-15.

Streubel, J., Blücher, C., Landgraf, A., & Boch, J. (2012). TAL effector RVD specificities and efficiencies. Nature Biotechnology, 30(7): 593.

Sun, Y., Jiao, G., Liu, Z., Zhang, X., Li, J., Guo, X., . . . Zhao, Y. (2017). Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in plant science, 8: 298.

Symington, L. S., & Gautier, J. (2011). Double-strand break end resection and repair pathway choice. Annual review of genetics, 45: 247-271.

Townsend, J. A., Wright, D. A., Winfrey, R. J., Fu, F., Maeder, M. L., Joung, J. K., & Voytas, D. F. (2009). High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature, 459(7245): 442.

Urnov, F. D., Miller, J. C., Lee, Y.-L., Beausejour, C. M., Rock, J. M., Augustus, S., . . . Holmes, M. C. (2005). Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435(7042): 646.

Voytas, D. F., & Gao, C. (2014). Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS biology, 12(6): e1001877.

Wah, D. A., Bitinaite, J., Schildkraut, I., & Aggarwal, A. K. (1998). Structure of FokI has implications for DNA cleavage. Proceedings of the National Academy of Sciences, 95(18): 10564-10569.

Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4): 910-918.

Wang, J., Friedman, G., Doyon, Y., Wang, N. S., Li, C. J., Miller, J. C., . . . Gregory, P. D. (2012). Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Research, 22(7): 1316-1326.

Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32(9): 947.

Wolfe, S. A., Nekludova, L., & Pabo, C. O. (2000). DNA recognition by Cys2His2 zinc finger proteins. Annual review of biophysics and biomolecular structure, 29(1): 183-212.

Xie, K., & Yang, Y. (2013). RNA-guided genome editing in plants using a CRISPR–Cas system. Molecular Plant, 6(6): 1975-1983.

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

Zaman, Q. U., Li, C., Cheng, H., & Hu, Q. (2018). Genome editing opens a new era of genetic improvement in polyploid


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