CRISPR/Cas9 and its Application in Plant Biotechnology



CRISPR/Cas9, genome editing, mutation, plant biotechnology


The clustered regularly interspaced short palindromic repeat-associated protein9 genome editing system (CRISPR/Cas9) was first discovered in prokaryotic organisms. Then, it was a widely used tool for eukaryotes. According to the other genome editing systems such as ZFNs and TALENs, CRISPR/Cas9 is much more preferred by researchers due to its simplicity and high efficiency. CRISPR /Cas9 vector systems have been developed for applying this technology to numerous plant species. In this study, we present a review of CRISPR/Cas9 system and its application in plant biotechnology.


Anders, C., Niewoehner, O., Duerst, A., & Jinek, M. (2014). Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513(7519), 569-573.

Andersson, A. F., & Banfield, J. F. (2008). Virus population dynamics and acquired virus resistance in natural microbial communities. Science, 320(5879), 1047-1050.

Arora, L., & Narula, A. (2017). Gene editing and crop improvement using CRISPR-Cas9 system. Frontiers in plant science, 8, 1932.

Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., & Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315(5819), 1709-1712.

Belhaj, K., Chaparro-Garcia, A., Kamoun, S., & Nekrasov, V. (2013). Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant methods, 9(1), 1-10.

Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., & Bonas, U. (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326(5959), 1509-1512.

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

Bogdanove, A. J., & Voytas, D. F. (2011). TAL effectors: customizable proteins for DNA targeting. Science, 333(6051), 1843-1846.

Borrelli, V. M., Brambilla, V., Rogowsky, P., Marocco, A., & Lanubile, A. (2018). The enhancement of plant disease resistance using CRISPR/Cas9 technology. Frontiers in plant science, 9, 1245.

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

Bragard, C., Caciagli, P., Lemaire, O., Lopez-Moya, J. J., MacFarlane, S., Peters, D., Peters, D., & Torrance, L. (2013). Status and prospects of plant virus control through interference with vector transmission. Annual review of phytopathology, 51, 177-201.

Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., Dickman, M.J., Makarova, K.S., Koonin, E.V., & Van Der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 321(5891), 960-964.

Butler, N. M., Atkins, P. A., Voytas, D. F., & 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.

Butler, N. M., Baltes, N. J., Voytas, D. F., & Douches, D. S. (2016). Geminivirus-mediated genome editing in potato (Solanum tuberosum L.) using sequence-specific nucleases. Frontiers in plant science, 7, 1045.

Cai, Y., Chen, L., Liu, X., Sun, S., Wu, C., Jiang, B., Han, T., & Hou, W. (2015). CRISPR/Cas9-mediated genome editing in soybean hairy roots. PLoS One, 10(8), e0136064.

Cencic, R., Miura, H., Malina, A., Robert, F., Ethier, S., Schmeing, T. M., & Pelletier, J. (2014). Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. PloS one, 9(10), e109213.

Čermák, T., Baltes, N. J., Čegan, R., Zhang, Y., & Voytas, D. F. (2015). High-frequency, precise modification of the tomato genome. Genome biology, 16(1), 1-15.

Chandrasekaran, J., Brumin, M., Wolf, D., Leibman, D., Klap, C., Pearlsman, M., Sherman, A., Arazi, T., & Gal‐On, A. (2016). Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology. Molecular plant pathology, 17(7), 1140-1153.

Chen, H., Choi, J., & Bailey, S. (2014). Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. Journal of Biological Chemistry, 289(19), 13284-13294.

Chen, K., Wang, Y., Zhang, R., Zhang, H., & Gao, C. (2019). CRISPR/Cas genome editing and precision plant breeding in agriculture. Annual review of plant biology, 70, 667-697.

Cho, S., Choe, D., Lee, E., Kim, S. C., Palsson, B., & Cho, B. K. (2018). High-level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS Synthetic Biology, 7(4), 1085-1094.

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

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

Cox, D. B., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing with CRISPR-Cas13. Science, 358(6366), 1019-1027.

Dangl, J. L., Horvath, D. M., & Staskawicz, B. J. (2013). Pivoting the plant immune system from dissection to deployment. Science, 341(6147), 746-751.

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

Demirel, S., Usta, M., & Demirel, F. (2020). CRISPR/Cas Technology in Resistance to Phytopathogens. European Journal of Science and Technology, (20), 693-702.

Dong, O. X., Yu, S., Jain, R., Zhang, N., Duong, P. Q., Butler, C., Li, Y., Lipzen, A., Martin, J.A., Barry, K.W., Schmutz, J., Tian, L., & Ronald, P. C. (2020). Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. Nature communications, 11(1), 1-10.

Fan, D., Liu, T., Li, C., Jiao, B., Li, S., Hou, Y., & Luo, K. (2015). Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation. Scientific reports, 5(1), 1-7.

FAO. (2016). State of Food Security and Nutrition in the World. Building Resilience for Peace and Food Security. Rome: FAO.

FAO. (2017). The Future of Food and Agriculture – Trends and Challenges. Rome, Italy.

Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S. L., & Gurr, S. J. (2012). Emerging fungal threats to animal, plant and ecosystem health. Nature, 484(7393), 186-194.

Gao, H., Gadlage, M. J., Lafitte, H. R., Lenderts, B., Yang, M., Schroder, M., Snopek, K., Peterson, D., Feigenbutz, L., Jones, S., St Clair, G., Rahe, M., Sanyour-Doyel, N., Peng, C., Wang, L., Young, J.K., Beatty, M., Dahlke, B., Hazebroek, J., Greene, T.W., Cigan, A.M., Chilcoat, N.D., & Meeley, R. B. (2020). Superior field performance of waxy corn engineered using CRISPR–Cas9. Nature biotechnology, 38(5), 579-581.

Garneau, J. E., Dupuis, M. È., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., & Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), 67-71.

Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences, 109(39), E2579-E2586.

Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017). Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage. Nature, 551(7681), 464-471.

Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., Lim, W. A., Weissman, J. S., & Qi, L. S. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154(2), 442–451.

Griggs, D., Smith, M. S., Rockström, J., Öhman, M. C., Gaffney, O., Glaser, G., Kanie, N., Noble, I., Steffen, W., & Shyamsundar, P. (2014). An integrated framework for sustainable development goals. Ecology and Society, 19(4).

Guan, X., Stege, J., Kim, M., Dahmani, Z., Fan, N., Heifetz, P., & Briggs, S. P. (2002). Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proceedings of the National Academy of Sciences, 99(20), 13296-13301.

Hanley-Bowdoin, L., Bejarano, E. R., Robertson, D., & Mansoor, S. (2013). Geminiviruses: masters at redirecting and reprogramming plant processes. Nature Reviews Microbiology, 11(11), 777-788.

Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., Ma, E., Witte, I. P., & Doudna, J. A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362(6416), 839-842.

Hayes, R. P., Xiao, Y., Ding, F., Van Erp, P. B., Rajashankar, K., Bailey, S., & Ke, A. (2016). Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli. Nature, 530(7591), 499-503.

Heler, R., Samai, P., Modell, J. W., Weiner, C., Goldberg, G. W., Bikard, D., & Marraffini, L. A. (2015). Cas9 specifies functional viral targets during CRISPR–Cas adaptation. Nature, 519(7542), 199-202.

Horvath, P., & Barrangou, R. (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science, 327(5962), 167-170.

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

Ito, Y., Nishizawa-Yokoi, A., Endo, M., Mikami, M., & 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.

Jaganathan, D., Ramasamy, K., Sellamuthu, G., Jayabalan, S., & Venkataraman, G. (2018). CRISPR for crop improvement: an update review. Frontiers in plant science, 9, 985.

Jensen, T. I., Mikkelsen, N. S., Gao, Z., Foßelteder, J., Pabst, G., Axelgaard, E., Laustsen, A., König, S., Reinisch., A., & Bak, R. O. (2021). Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi. Genome Research, 31(11), 2120-2130.

Jia, H., & Wang, N. (2014). Targeted genome editing of sweet orange using Cas9/sgRNA. PloS one, 9(4), e93806.

Jia, H., Zhang, Y., Orbović, V., Xu, J., White, F. F., Jones, J. B., & Wang, N. (2017). Genome editing of the disease susceptibility gene Cs LOB 1 in citrus confers resistance to citrus canker. Plant biotechnology journal, 15(7), 817-823.

Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B., & 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-e188.

Jiang, F., Zhou, K., Ma, L., Gressel, S., & Doudna, J. A. (2015). A Cas9–guide RNA complex preorganized for target DNA recognition. Science, 348(6242), 1477-1481.

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, F., & Doudna, J. A. (2017). CRISPR–Cas9 structures and mechanisms. Annual review of biophysics, 46, 505-529.

Jiang, Y., Qian, F., Yang, J., Liu, Y., Dong, F., Xu, C., Sun, B., Chen, B., Xu, X., Li, Y., Wang, R., & Yang, S. (2017). CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nature communications, 8, 15179.

Jin, S., Fei, H., Zhu, Z., Luo, Y., Liu, J., Gao, S., Zhang, F., Chen, Y. H., Wang, Y., & Gao, C. (2020). Rationally designed APOBEC3B cytosine base editors with improved specificity. Molecular cell, 79(5), 728-740.

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.

Jinek, M., Jiang, F., Taylor, D. W., Sternberg, S. H., Kaya, E., Ma, E., & Doudna, J. A. (2014). Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 343(6176).

Josephs, E. A., Kocak, D. D., Fitzgibbon, C. J., McMenemy, J., Gersbach, C. A., & Marszalek, P. E. (2015). Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic acids research, 43(18), 8924-8941.

Jung, H., Lee, A., Jo, S. H., Park, H. J., Jung, W. Y., Kim, H. S., Lee, H. J., Jeong, S. G., Kim, Y. S., & Cho, H. S. (2021). Nitrogen Signaling Genes and SOC1 Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage (Brassica rapa L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple BrSOC1 Homologs. International journal of molecular sciences, 22(9), 4631.

Kang, B. C., Yun, J. Y., Kim, S. T., Shin, Y., Ryu, J., Choi, M., Woo, J. W., & Kim, J. S. (2018). Precision genome engineering through adenine base editing in plants. Nature Plants, 4(7), 427-431.

Kapusi, E., Corcuera-Gómez, M., Melnik, S., & Stoger, E. (2017). Heritable genomic fragment deletions and small indels in the putative ENGase gene induced by CRISPR/Cas9 in barley. Frontiers in plant science, 8, 540.

Karvelis, T., Gasiunas, G., Miksys, A., Barrangou, R., Horvath, P., & Siksnys, V. (2013). crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA biology, 10(5), 841-851.

Kaur, N., Alok, A., Kaur, N., Pandey, P., Awasthi, P., & Tiwari, S. (2018). CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome. Functional & integrative genomics, 18(1), 89-99.

Kim, D., Alptekin, B., & Budak, H. (2018). CRISPR/Cas9 genome editing in wheat. Functional & integrative genomics, 18(1), 31-41.

Kloepper, J. W., Ryu, C. M., & Zhang, S. (2004). Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology, 94(11), 1259-1266.

Knight, S. C., Xie, L., Deng, W., Guglielmi, B., Witkowsky, L. B., Bosanac, L., & Tjian, R. (2015). Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science, 350(6262), 823-826.

Knoll, A., Fauser, F., & Puchta, H. (2014). DNA recombination in somatic plant cells: mechanisms and evolutionary consequences. Chromosome Research, 22(2), 191-201.

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

Kumar, V., & Jain, M. (2015). The CRISPR–Cas system for plant genome editing: advances and opportunities. Journal of experimental botany, 66(1), 47-57.

Langner, T., Kamoun, S., & Belhaj, K. (2018). CRISPR crops: plant genome editing toward disease resistance. Annual review of phytopathology, 56, 479-512.

Li, J. F., Norville, J. E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G. M., & Sheen, J. (2013). Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology, 31(8), 688-691.

Li, C., Zong, Y., Wang, Y., Jin, S., Zhang, D., Song, Q., Zhang, R., & Gao, C. (2018). Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion. Genome biology, 19(1), 1-9.

Li, R., Liu, C., Zhao, R., Wang, L., Chen, L., Yu, W., Zhang, S., Sheng, J., & Shen, L. (2019). CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance. BMC plant biology, 19(1), 1-13.

Li, C., Zhang, R., Meng, X., Chen, S., Zong, Y., Lu, C., Qiu, J. L., Chen, Y. H., Li, J., & Gao, C. (2020). Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nature biotechnology, 38(7), 875-882.

Lieber, M. R. (2010). The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual review of biochemistry, 79, 181-211.

Liu, W., Rudis, M. R., Peng, Y., Mazarei, M., Millwood, R. J., Yang, J. P., & Stewart Jr, C. N. (2014). Synthetic TAL effectors for targeted enhancement of transgene expression in plants. Plant biotechnology journal, 12(4), 436-446.

Ma, H., Tu, L. C., Naseri, A., Huisman, M., Zhang, S., Grunwald, D., & Pederson, T. (2016). CRISPR-Cas9 nuclear dynamics and target recognition in living cells. Journal of Cell Biology, 214(5), 529-537.

Maeder, M. L., Thibodeau-Beganny, S., Osiak, A., Wright, D. A., Anthony, R. M., Eichtinger, M., & Joung, J. K. (2008). Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Molecular cell, 31(2), 294-301.

Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J. M., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., van der Oost, J., Backofen, R., & Koonin, E. V. (2015). An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology, 13(11), 722-736.

Makarova, K. S., Wolf, Y. I., & Koonin, E. V. (2018). Classification and nomenclature of CRISPR-Cas systems: where from here?. The CRISPR Journal, 1(5), 325-336.

Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., & Church, G. M. (2013a). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology, 31(9), 833-838.

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

Malnoy, M., Viola, R., Jung, M. H., Koo, O. J., Kim, S., Kim, J. S., Velasco, R., & Nagamangala Kanchiswamy, C. (2016). DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Frontiers in plant science, 7, 1904.

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

Matres, J. M., Hilscher, J., Datta, A., Armario-Nájera, V., Baysal, C., He, W., & Slamet-Loedin, I. H. (2021). Genome editing in cereal crops: an overview. Transgenic research, 1-38.

Maxson-Stein, K., He, S. Y., Hammerschmidt, R., & Jones, A. L. (2002). Effect of treating apple trees with acibenzolar-S-methyl on fire blight and expression of pathogenesis-related protein genes. Plant disease, 86(7), 785-790.

Michno, J. M., Wang, X., Liu, J., Curtin, S. J., Kono, T. J., & 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, 6(4), 243-252.

Morbitzer, R., Römer, P., Boch, J., & Lahaye, T. (2010). Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proceedings of the National Academy of Sciences, 107(50), 21617-21622.

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

Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D., & Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature biotechnology, 31(8), 691-693.

Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D., & Kamoun, S. (2017). Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Scientific reports, 7(1), 1-6.

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.

Nishitani, C., Hirai, N., Komori, S., Wada, M., Okada, K., Osakabe, K., Yamamoto, T., & Osakabe, Y. (2016). Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific reports, 6(1), 1-8.

O’Connell, M. R., Oakes, B. L., Sternberg, S. H., East-Seletsky, A., Kaplan, M., & Doudna, J. A. (2014). Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature, 516(7530), 263-266.

O’Connell, M. R. (2019). Molecular mechanisms of RNA targeting by Cas13-containing type VI CRISPR–Cas systems. Journal of molecular biology, 431(1), 66-87.

Oliva, R., Ji, C., Atienza-Grande, G., Huguet-Tapia, J. C., Perez-Quintero, A., Li, T., Eom, J. S., Li, C., Nguyen, H., Liu, B., Auguy, F., Sciallano, C., Luu, V. T., Dossa, G. S., Cunnac, S., Schmidt, S. M., Slamet-Loedin, I. H., Vera Cruz, C., Szurek, B., Frommer, W. B., White, F. F., & Yang, B. (2019). Broad-spectrum resistance to bacterial blight in rice using genome editing. Nature biotechnology, 37(11), 1344-1350.

Palermo, G., Miao, Y., Walker, R. C., Jinek, M., & McCammon, J. A. (2016). Striking plasticity of CRISPR-Cas9 and key role of non-target DNA, as revealed by molecular simulations. ACS central science, 2(10), 756-763.

Pan, C., Ye, L., Qin, L., Liu, X., He, Y., Wang, J., Chen, L., & Lu, G. (2016). CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific reports, 6(1), 1-9.

Paszkowski, J., Baur, M., Bogucki, A., & Potrykus, I. (1988). Gene targeting in plants. The EMBO journal, 7(13), 4021-4026.

Peng, A., Chen, S., Lei, T., Xu, L., He, Y., Wu, L., Yao, L., & Zou, X. (2017). Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene Cs LOB 1 promoter in citrus. Plant biotechnology journal, 15(12), 1509-1519.

Pradhanang, P. M., Ji, P., Momol, M. T., Olson, S. M., Mayfield, J. L., & Jones, J. B. (2005). Application of acibenzolar-S-methyl enhances host resistance in tomato against Ralstonia solanacearum. Plant disease, 89(9), 989-993.

Price, A. A., Sampson, T. R., Ratner, H. K., Grakoui, A., & Weiss, D. S. (2015). Cas9-mediated targeting of viral RNA in eukaryotic cells. Proceedings of the National Academy of Sciences, 112(19), 6164-6169.

Price, A. A., Grakoui, A., & Weiss, D. S. (2016). Harnessing the prokaryotic adaptive immune system as a eukaryotic antiviral defense. Trends in microbiology, 24(4), 294-306.

Puchta, H., Dujon, B., & Hohn, B. (1993). Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic acids research, 21(22), 5034-5040.

Puchta, H. (2005). The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. Journal of experimental botany, 56(409), 1-14.

Puchta, H. (2017). Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Current opinion in plant biology, 36, 1-8.

Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173-1183.

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

Ren, C., Liu, X., Zhang, Z., Wang, Y., Duan, W., Li, S., & Liang, Z. (2016). CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Scientific reports, 6(1), 1-9.

Ricroch, A., Clairand, P., & Harwood, W. (2017). Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture. Emerging Topics in Life Sciences, 1(2), 169-182.

Rohs, R., Jin, X., West, S. M., Joshi, R., Honig, B., & Mann, R. S. (2010). Origins of specificity in protein-DNA recognition. Annual review of biochemistry, 79, 233-269.

Rutkauskas, M., Sinkunas, T., Songailiene, I., Tikhomirova, M. S., Siksnys, V., & Seidel, R. (2015). Directional R-loop formation by the CRISPR-Cas surveillance complex cascade provides efficient off-target site rejection. Cell reports, 10(9), 1534-1543.

Sauer, N. J., Narváez-Vásquez, J., Mozoruk, J., Miller, R. B., Warburg, Z. J., Woodward, M. J., & Gocal, G. F. (2016). Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant physiology, 170(4), 1917-1928.

Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P., & Siksnys, V. (2011). The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic acids research, 39(21), 9275-9282.

Schaart, J. G., van de Wiel, C. C., & Smulders, M. J. (2021). Genome editing of polyploid crops: prospects, achievements and bottlenecks. Transgenic Research, 1-15.

Scheben, A., Wolter, F., Batley, J., Puchta, H., & Edwards, D. (2017). Towards CRISPR/Cas crops–bringing together genomics and genome editing. New Phytologist, 216(3), 682-698.

Scheben, A., & Edwards, D. (2018) Bottlenecks for genome-edited crops on the road from lab to farm. Genome Biol, 19, 178.

Schornack, S., Moscou, M. J., Ward, E. R., & Horvath, D. M. (2013). Engineering plant disease resistance based on TAL effectors. Annual review of phytopathology, 51, 383-406.

Schultz, B. (2018). Contribution of water management and flood protection to food security and sustainable development of coastal and deltaic areas. Irrigation and Drainage, 67(1), 123-135.

Sen, Y., van der Wolf, J., Visser, R. G., & van Heusden, S. (2015). Bacterial canker of tomato: current knowledge of detection, management, resistance, and interactions. Plant Disease, 99(1), 4-13.

Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., & Gao, C. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nature biotechnology, 31(8), 686-688.

Shan, Q., Zhang, Y., Chen, K., Zhang, K., & Gao, C. (2015). Creation of fragrant rice by targeted knockout of the Os BADH 2 gene using TALEN technology. Plant biotechnology journal, 13(6), 791-800.

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. (2017). Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nature biotechnology, 35(5), 441–443.

Shukla, V. K., Doyon, Y., Miller, J. C., DeKelver, R. C., Moehle, E. A., Worden, S. E., & Urnov, F. D. (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459(7245), 437-441.

Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A., & Ha, T. (2016). Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nature communications, 7(1), 1-8.

Sobiczewski, P. (2008). Bacterial diseases of plants: Epidemiology, diagnostics and control. Zemdirbyste, 95, 151-157.

Steduto, P., Schultz, B., Unver, O., Ota, S., Vallee, D., Kulkarni, S., & Dagnino‐Johns Garcia, M. (2018). Food security by optimal use of water: synthesis of the 6th and 7th world water forums and developments since then. Irrigation and Drainage, 67(3), 327-344.

Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507(7490), 62-67.

Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015). Conformational control of DNA target cleavage by CRISPR–Cas9. Nature, 527(7576), 110-113.

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

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

Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K., & Cigan, A. M. (2016). Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nature communications, 7(1), 1-7.

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

Szczelkun, M. D., Tikhomirova, M. S., Sinkunas, T., Gasiunas, G., Karvelis, T., Pschera, P., & Seidel, R. (2014). Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proceedings of the National Academy of Sciences, 111(27), 9798-9803.

Taghbalout, A., Du, M., Jillette, N., Rosikiewicz, W., Rath, A., Heinen, C. D., Li, S., & Cheng, A. W. (2019). Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways. Nature communications, 10(1), 1-12.

Tilman, D., Balzer, C., Hill, J., & Befort, B. L. (2011). Global food demand and the sustainable intensification of agriculture. Proceedings of the national academy of sciences, 108(50), 20260-20264.

Tufan, F., & Keleş, E.N. (2019). Genome Editing Technologies and its Applications in Plants. Haliç Üniversitesi Fen Bilimleri Dergisi, 2(1), 113-133.

Van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M., & Brouns, S. J. (2009). CRISPR-based adaptive and heritable immunity in prokaryotes. Trends in biochemical sciences, 34(8), 401-407.

van Regenmortel, M. H., & Mahy, B. W. (Eds.). (2009). Desk encyclopedia of plant and fungal virology. Academic Press.

Voytas, D. F. (2013). Plant genome engineering with sequence-specific nucleases. Annual review of plant biology, 64, 327-350.

Wagh, S. G., & Pohare, M. B. (2019). Current and future prospects of plant breeding with CRISPR/Cas. Current Journal of Applied Science and Technology, 38(3), 1-17.

Waltz, E. (2018). With a free pass, CRISPR-edited plants reach market in record time. Nature biotechnology, 36(1), 6-8.

Wang, M., Wang, G., Ji, J., & Wang, J. (2009). The effect of pds gene silencing on chloroplast pigment composition, thylakoid membrane structure and photosynthesis efficiency in tobacco plants. Plant Science, 177(3), 222-226.

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), 947-951.

Wang, S., Zhang, S., Wang, W., Xiong, X., Meng, F., & Cui, X. (2015). Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant cell reports, 34(9), 1473-1476.

Wang, F., Wang, C., Liu, P., Lei, C., Hao, W., Gao, Y., Liu, Y. G., & Zhao, K. (2016a). Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PloS one, 11(4), e0154027.

Wang, Y., Liu, X., Ren, C., Zhong, G. Y., Yang, L., Li, S., & Liang, Z. (2016b). Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome. BMC plant biology, 16(1), 1-7.

Wang, Y., Zong, Y., & Gao, C. (2017). Targeted mutagenesis in hexaploid bread wheat using the TALEN and CRISPR/Cas systems. In Wheat Biotechnology (pp. 169-185). Humana Press, New York, NY.

Wang, X., Tu, M., Wang, D., Liu, J., Li, Y., Li, Z., Wang, Y., & Wang, X. (2018). CRISPR/Cas9‐mediated efficient targeted mutagenesis in grape in the first generation. Plant biotechnology journal, 16(4), 844-855.

Wang, C., Wang, G., Gao, Y., Lu, G., Habben, J. E., Mao, G., Chen, G., Wang, J., Yang, F., Zhao, X., Zhang, J., Mo, H., Qu, P., Liu, J., & Greene, T. W. (2020). A cytokinin-activation enzyme-like gene improves grain yield under various field conditions in rice. Plant molecular biology, 102(4), 373-388.

Woo, J. W., Kim, J., Kwon, S. I., Corvalán, 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 CRISPR-Cas9 ribonucleoproteins. Nature biotechnology, 33(11), 1162-1164.

Wright, D. A., Townsend, J. A., Winfrey Jr, R. J., Irwin, P. A., Rajagopal, J., Lonosky, P. M., & Voytas, D. F. (2005). High‐frequency homologous recombination in plants mediated by zinc‐finger nucleases. The Plant Journal, 44(4), 693-705.

Wu, X., Scott, D. A., Kriz, A. J., Chiu, A. C., Hsu, P. D., Dadon, D. B., & Sharp, P. A. (2014). Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nature biotechnology, 32(7), 670-676.

Wu, T., Ali, A., Wang, J., Song, J., Fang, Y., Zhou, T., Luo, Y., Zhang, H., Chen, X., Liao, Y., Liu, Y., Xu, P., & Wu, X. (2021). A homologous gene of OsREL2/ASP1, ASP-LSL regulates pleiotropic phenotype including long sterile lemma in rice. BMC Plant Biology, 21(1), 1-15.

Xu, X., & Qi, L. S. (2019). A CRISPR–dCas toolbox for genetic engineering and synthetic biology. Journal of molecular biology, 431(1), 34-47.

Xu, Z., Xu, X., Gong, Q., Li, Z., Li, Y., Wang, S., Yang, Y., Ma, W., Liu, L., Zhu, B., Zou, L., & Chen, G. (2019). Engineering broad-spectrum bacterial blight resistance by simultaneously disrupting variable TALE-binding elements of multiple susceptibility genes in rice. Molecular plant, 12(11), 1434-1446.

Xu, Y., Lin, Q., Li, X., Wang, F., Chen, Z., Wang, J., Li, W., Fan, F., Tao, Y., Jiang, Y., Wei, X., Zhang, R., Zhu, Q. H., Bu, Q., Yang, J., & Gao, C. (2021). Fine-tuning the amylose content of rice by precise base editing of the Wx gene. Plant biotechnology journal, 19(1), 11–13.

Yuste-Lisbona, F. J., Fernández-Lozano, A., Pineda, B., Bretones, S., Ortíz-Atienza, A., García-Sogo, B., Müller, N. A., Angosto, T., Capel, J., Moreno, V., Jiménez-Gómez, J. M., & Lozano, R. (2020). ENO regulates tomato fruit size through the floral meristem development network. Proceedings of the National Academy of Sciences, 117(14), 8187-8195.

Zaidi, S. S. E. A., Tashkandi, M., Mansoor, S., & Mahfouz, M. M. (2016). Engineering plant immunity: using CRISPR/Cas9 to generate virus resistance. Frontiers in plant science, 7, 1673.

Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou, F., Feng, Z., Mao, Y., Yang, L., Zhang, H., Xu, N., & 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.

Zhang, Y., Liang, Z., Zong, Y., Wang, Y., Liu, J., Chen, K., Qiu, J. L., & Gao, C. (2016). Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature communications, 7(1), 1-8.

Zhang, Y., Bai, Y., Wu, G., Zou, S., Chen, Y., Gao, C., & Tang, D. (2017). Simultaneous modification of three homoeologs of Ta EDR 1 by genome editing enhances powdery mildew resistance in wheat. The Plant Journal, 91(4), 714-724.

Zhang, Z., Hua, L., Gupta, A., Tricoli, D., Edwards, K. J., Yang, B., & Li, W. (2019). Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing. Plant biotechnology journal, 17(8), 1623-1635.

Zhang, Y., Held, M. A., Kaur, D., & Showalter, A. M. (2021). CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis. BMC Plant Biology, 21(1), 1-16.

Zheng, Z., Appiano, M., Pavan, S., Bracuto, V., Ricciardi, L., Visser, R. G., Wolters, A. M. A., & Bai, Y. (2016). Genome-wide study of the tomato SlMLO gene family and its functional characterization in response to the powdery mildew fungus Oidium neolycopersici. Frontiers in plant science, 7, 380.

Zheng, Y., Li, J., Wang, B., Han, J., Hao, Y., Wang, S., Ma, X., Yang, S., Ma, L., Yi, L., & Peng, W. (2020). Endogenous type I CRISPR-Cas: from foreign DNA defense to prokaryotic engineering. Frontiers in bioengineering and biotechnology, 8, 62.




How to Cite

İlhan, E., Kasapoğlu, A. G. ., Muslu, S. ., Macit, M. ., Sezer, B. ., Mevlütoğulları, A. ., Güler, D., Aydın, M., Ekşi, F. ., & Aydın, M. . (2021). CRISPR/Cas9 and its Application in Plant Biotechnology. Natural Products and Biotechnology, 1(2), 118–143. Retrieved from



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