Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins
Abstract
Key message Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
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Effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult due to the large size of Cas9 protein (~160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA.
“However, due to the large size of Cas9 protein (~ 160 kD), the positive charge of Cas9 protein and the strong negative charge of sgRNA, the effective delivery of Cas9/sgRNA ribonucleoprotein complexes (RNPs) can be difficult [88–91].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [89]
Cas9 protein from S. pyogenes has molecular weight about 160 KD, larger than most proteins.
“The molecular weight of the commonly used Cas9 protein from streptococcus pyogenes is about 160 KD, larger than that of the most proteins [89].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [89]
First obstacle: effective encapsulation of editing tools in delivery vectors is hindered by large size and different charge properties of Cas9 protein, mRNA and donor DNA.
“The first obstacle is effective encapsulation of the editing tools in delivery vectors due to the large size and different charge properties of Cas9 protein, mRNA and donor DNA.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [89]
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Development and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762 · see citing works
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013) · Trends in Biotechnology · doi:10.1016/j.tibtech.2013.04.004 · Cited by 3,820 · see citing works
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153 · see citing works
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937 · see citing works
Bae, S., Park, J., & Kim, J.-S. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu048 · Cited by 2,339 · see citing works
Self-citationWang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014) · Nature Biotechnology · doi:10.1038/nbt.2969 · Cited by 2,072 · see citing works
Targeted genome modification of crop plants using a CRISPR-Cas system
Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J., Qiu, J., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2650 · Cited by 1,965 · see citing works
Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013) · Nature Biotechnology · doi:10.1038/nbt.2507 · Cited by 1,863 · see citing works
Self-citationKim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J.-S. (2014) · Genome Research · doi:10.1101/gr.171322.113 · Cited by 1,856 · see citing works
Self-citationGenome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt135 · Cited by 1,566 · see citing works
Li, J., Norville, J. E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G. M., & Sheen, J. (2013) · Nature Biotechnology · doi:10.1038/nbt.2654 · Cited by 1,499 · see citing works
Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases
Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., & Kim, J.-S. (2014) · Genome Research · doi:10.1101/gr.162339.113 · Cited by 1,356 · see citing works
Self-citationJiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B., & Weeks, D. P. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt780 · Cited by 1,250 · see citing works
DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins
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) · Nature Biotechnology · doi:10.1038/nbt.3389 · Cited by 1,219 · see citing works
Self-citationA guide to genome engineering with programmable nucleases
Kim, S., & Kim, J. (2014) · Nature Reviews Genetics · doi:10.1038/nrg3686 · Cited by 1,162 · see citing works
Self-citationHigh-efficiency TALEN-based gene editing produces disease-resistant rice
Li, T., Liu, B., Spalding, M. H., Weeks, D. P., & Yang, B. (2012) · Nature Biotechnology · doi:10.1038/nbt.2199 · Cited by 1,149 · see citing works
Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease
Nekrasov, V., Staskawicz, B. J., Weigel, D., Jones, J. D. G., & Kamoun, S. (2013) · Nature Biotechnology · doi:10.1038/nbt.2655 · Cited by 1,123 · see citing works
Efficient genome editing in plants using a CRISPR/Cas system
Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, D., Wei, P., Cao, F., Zhu, S., Zhang, F., Mao, Y., et al. (2013) · Cell Research · doi:10.1038/cr.2013.114 · Cited by 1,070 · see citing works
An improved zinc-finger nuclease architecture for highly specific genome editing
Miller, J. C., Holmes, M. C., Wang, J., Guschin, D. Y., Lee, Y.-L., Rupniewski, I., Beausejour, C. M., Waite, A. J., Wang, N. S., Kim, K. A., et al. (2007) · Nature Biotechnology · doi:10.1038/nbt1319 · Cited by 1,051 · see citing works
Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases
Perez, E. E., Wang, J., Miller, J. C., Jouvenot, Y., Kim, K. A., Liu, O., Wang, N., Lee, G., Bartsevich, V. V., Lee, Y.-L., et al. (2008) · Nature Biotechnology · doi:10.1038/nbt1410 · Cited by 999 · see citing works
Self-citationTargeted mutagenesis in rice using CRISPR-Cas system
Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., Wan, J., Gu, H., & Qu, L. (2013) · Cell Research · doi:10.1038/cr.2013.123 · Cited by 908 · see citing works
RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System
Xie, K., & Yang, Y. (2013) · Molecular Plant · doi:10.1093/mp/sst119 · Cited by 806 · see citing works
DNA Double-Strand Break Repair: All's Well that Ends Well
Wyman, C., & Kanaar, R. (2006) · Annual Review of Genetics · doi:10.1146/annurev.genet.40.110405.090451 · Cited by 806 · see citing works
Svitashev, S. K., Young, J. K., Schwartz, C., Gao, H., Falco, S. C., & Cigan, A. M. (2015) · PLANT PHYSIOLOGY · doi:10.1104/pp.15.00793 · Cited by 739 · see citing works
Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System
Liang, Z., Zhang, K., Chen, K., & Gao, C. (2013) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2013.12.001 · Cited by 662 · see citing works
Hybrid genes in the analysis of transformation conditions
Negrutiu, I., Shillito, R. D., Potrykus, I., Biasini, M. G., & Florin, S. A. L. A. (1987) · Plant Molecular Biology · doi:10.1007/bf00015814 · Cited by 524 · see citing works
Engineering polydactyl zinc-finger transcription factors
Beerli, R. R., & Barbas, C. F. (2002) · Nature Biotechnology · doi:10.1038/nbt0202-135 · Cited by 495 · see citing works
Fok I dimerization is required for DNA cleavage
Bitinaite, J., Wah, D. A., Aggarwal, A. K., & Schildkraut, I. (1998) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.95.18.10570 · Cited by 490 · see citing works
Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites
Park, J., Bae, S., & Kim, J. (2015) · Bioinformatics · doi:10.1093/bioinformatics/btv537 · Cited by 483 · see citing works
Self-citationTargeted genome editing in human cells with zinc finger nucleases constructed via modular assembly
Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., & Kim, J.-S. (2009) · Genome Research · doi:10.1101/gr.089417.108 · Cited by 466 · see citing works
Self-citationMicrohomology-based choice of Cas9 nuclease target sites
Bae, S., Kweon, J., Kim, H. S., & Kim, J. (2014) · Nature Methods · doi:10.1038/nmeth.3015 · Cited by 380 · see citing works
Self-citationCRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum
Gao, J., Wang, G., Ma, S., Xie, X., Wu, X., Zhang, X., Wu, Y., Zhao, P., & Xia, Q. (2014) · Plant Molecular Biology · doi:10.1007/s11103-014-0263-0 · Cited by 340 · see citing works
Wilkinson, J. Q., & Crawford, N. M. (1993) · Molecular and General Genetics MGG · doi:10.1007/bf00281630 · Cited by 339 · see citing works
Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice
Aida, T., Chiyo, K., Usami, T., Ishikubo, H., Imahashi, R., Wada, Y., Tanaka, K. F., Sakuma, T., Yamamoto, T., & Tanaka, K. (2015) · Genome biology · doi:10.1186/s13059-015-0653-x · Cited by 296 · see citing works
Vandenbussche, M., Zethof, J., Royaert, S., Weterings, K., & Gerats, T. (2004) · The Plant Cell · doi:10.1105/tpc.019166 · Cited by 220 · see citing works
Morton, J. J., Davis, M. W., Jørgensen, E. M., & Carroll, D. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0605633103 · Cited by 181 · see citing works
Hyun, Y., Kim, J., Cho, S. W., Choi, Y., Kim, J., & Coupland, G. (2014) · Planta · doi:10.1007/s00425-014-2180-5 · Cited by 167 · see citing works
Self-citationJiang, W., Yang, B., & Weeks, D. P. (2014) · PLoS ONE · doi:10.1371/journal.pone.0099225 · Cited by 163 · see citing works
A model system for comparative research: Petunia
Gerats, T., & Vandenbussche, M. (2005) · Trends in Plant Science · doi:10.1016/j.tplants.2005.03.005 · Cited by 147 · see citing works
TALENs: Customizable Molecular DNA Scissors for Genome Engineering of Plants
Chen, K., & Gao, C. (2013) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2013.03.009 · Cited by 114 · see citing works
TALENs and ZFNs are associated with different mutation signatures
Kim, Y., Kweon, J., & Kim, J. (2013) · Nature Methods · doi:10.1038/nmeth.2364 · Cited by 97 · see citing works
Self-citationPark, C. Y., Krishnan, A., Zhu, Q., Wong, A. K., Lee, Y.-S., & Troyanskaya, O. G. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu786 · Cited by 96 · see citing works
Non-GMO genetically edited crop plants
Kanchiswamy, C. N., Malnoy, M., Velasco, R., Kim, J., & Viola, R. (2015) · Trends in biotechnology · doi:10.1016/j.tibtech.2015.04.002 · Cited by 73 · see citing works
Self-citationNon-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases
Luo, S., Li, J., Stoddard, T., Baltes, N. J., Demorest, Z. L., Clasen, B. M., Coffman, A. P., Retterath, A., Mathis, L., Voytas, D. F., et al. (2015) · Molecular Plant · doi:10.1016/j.molp.2015.05.012 · Cited by 63 · see citing works
Protoplast isolation and plant regeneration of different genotypes of Petunia and Calibrachoa
Meyer, L., Serek, M., & Winkelmann, T. (2009) · Plant Cell Tissue and Organ Culture (PCTOC) · doi:10.1007/s11240-009-9572-4 · Cited by 45 · see citing works
van Houwelingen, A. M. M. L., Souer, E., Mol, J. N. M., & Koes, R. E. (1999) · The Plant Cell · doi:10.1105/tpc.11.7.1319 · Cited by 39 · see citing works
Petunia as a Model System for the Genetics and Evolution of Pollination Syndromes
Gübitz, T., Hoballah, M. E., Dell’Olivo, A., & Kuhlemeier, C. J. (2008) · Springer eBooks · doi:10.1007/978-0-387-84796-2_2 · Cited by 38 · see citing works
Zhao, X., Nie, X.-L., & Xiao, X.-G. (2013) · PLoS ONE · doi:10.1371/journal.pone.0074678 · Cited by 34 · see citing works
Meyer, P. (2001) · Current Opinion in Plant Biology · doi:10.1016/s1369-5266(00)00200-4 · Cited by 30 · see citing works
Systematic silencing of a tobacco nitrate reductase transgene in lettuce (Lactuca sativa L.)
Dubois, V., Botton, E., Meyer, C., Rieu, A., Bedu, M., Maisonneuve, B., & Mazier, M. (2005) · Journal of Experimental Botany · doi:10.1093/jxb/eri230 · Cited by 25 · see citing works
Transformation and Regeneration of Petunia
Conner, A. J., Albert, N. W., & Deroles, S. C. (2008) · Springer eBooks · doi:10.1007/978-0-387-84796-2_19 · Cited by 24 · see citing works
Plant regeneration from petal protoplast culture ofPetunia hybrida
Oh, M., & Kim, S. (1994) · Plant Cell Tissue and Organ Culture (PCTOC) · doi:10.1007/bf00046084 · Cited by 17 · see citing works
Salanoubat, M., & Ha, D. B. D. (1993) · Gene · doi:10.1016/0378-1119(93)90557-j · Cited by 15 · see citing works
Vaucheret, H., Palauqui, J., Mourrain, P., & Elmayan, T. (1997) · Euphytica · doi:10.1023/a:1002996818918 · Cited by 4 · see citing works
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Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities
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The emerging and uncultivated potential of CRISPR technology in plant science
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Self-citationCRISPR-Cas9 system: A new-fangled dawn in gene editing
Gupta, D., Bhattacharjee, O., Mandal, D., Sen, M. K., Dey, D., Dasgupta, A., Kazi, T. A., Gupta, R. K., Sinharoy, S., Acharya, K., et al. (2019) · Life Sciences · doi:10.1016/j.lfs.2019.116636 · Cited by 332 · see citing works
Plant genome editing with TALEN and CRISPR
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Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering
Wada, N., Ueta, R., Osakabe, Y., & Osakabe, K. (2020) · BMC Plant Biology · doi:10.1186/s12870-020-02385-5 · Cited by 271 · see citing works
Liang, Z., Chen, K., Zhang, Y., Liu, J., Yin, K., Qiu, J., & Gao, C. (2018) · Nature Protocols · doi:10.1038/nprot.2017.145 · Cited by 238 · see citing works
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Mushtaq, M., Dar, A. A., Skalický, M., Tyagi, A., Bhagat, N., Basu, U., Bhat, B. A., Zaid, A., Ali, S., Dar, T. U. H., et al. (2021) · Genes · doi:10.3390/genes12060797 · Cited by 100 · see citing works
CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments
Montecillo, J. A. V., Chu, L. L., & Bae, H. (2020) · Agronomy · doi:10.3390/agronomy10071033 · Cited by 94 · see citing works
Su, H., Wang, Y., Xu, J. H., Omar, A. A., Grosser, J. W., Ćalović, M., Zhang, L., Yu, F., Vakulskas, C. A., & Wang, N. (2023) · Nature Communications · doi:10.1038/s41467-023-39714-9 · Cited by 87 · see citing works
Transgene-free Genome Editing in Plants
Gu, X., Liu, L., & Zhang, H. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.805317 · Cited by 86 · see citing works
Genome editing and plant transformation of solanaceous food crops
Eck, J. M. V. (2017) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2017.07.012 · Cited by 85 · see citing works
Corte, L. E.-D., Mahmoud, L. M., Moraes, T. D. S., Mou, Z., Grosser, J. W., & Dutt, M. (2019) · Plants · doi:10.3390/plants8120601 · Cited by 85 · see citing works
Genome editing in crop improvement: Present scenario and future prospects
Miglani, G. S. (2017) · Journal of Crop Improvement · doi:10.1080/15427528.2017.1333192 · Cited by 84 · see citing works
Najafi, S., Bertini, E., Erica D’Incà, Fasoli, M., & Zenoni, S. (2022) · Horticulture Research · doi:10.1093/hr/uhac240 · Cited by 83 · see citing works
New traits in crops produced by genome editing techniques based on deletions
van de Wiel, C. C. M., Schaart, J. G., Lotz, L. A. P., & Smulders, M. J. M. (2017) · Plant Biotechnology Reports · doi:10.1007/s11816-017-0425-z · Cited by 80 · see citing works
Targeted modification of plant genomes for precision crop breeding
Hilscher, J., Buerstmayr, H., & Stöger, E. (2016) · Biotechnology Journal · doi:10.1002/biot.201600173 · Cited by 79 · see citing works
Fan, Y., Xin, S., Dai, X., Yang, X., Huang, H., & Hua, Y. (2020) · Industrial Crops and Products · doi:10.1016/j.indcrop.2020.112146 · Cited by 77 · see citing works
PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts
Sant’Ana, R. R. A., Caprestano, C. A., Nodari, R. O., & Agapito-Tenfen, S. Z. (2020) · Genes · doi:10.3390/genes11091029 · Cited by 78 · see citing works
Xia, X., Cheng, X., Li, R., Yao, J., Li, Z., & Cheng, Y. (2021) · Theoretical and Applied Genetics · doi:10.1007/s00122-021-03874-3 · Cited by 78 · see citing works
Li, Q., Sapkota, M., & van der Knaap, E. (2020) · Horticulture Research · doi:10.1038/s41438-020-0258-8 · Cited by 73 · see citing works
DNA-free genome editing methods for targeted crop improvement
Kanchiswamy, C. N. (2016) · Plant Cell Reports · doi:10.1007/s00299-016-1982-2 · Cited by 69 · see citing works
Genome editing technology and application in soybean improvement
Bao, A., Zhang, C., Huang, Y., Chen, H., Zhou, X., & Cao, D. (2020) · Oil Crop Science · doi:10.1016/j.ocsci.2020.03.001 · Cited by 68 · see citing works
Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)
Poovaiah, C. R., Phillips, L., Geddes, B., Reeves, C., Sorieul, M., & Thorlby, G. J. (2021) · BMC Plant Biology · doi:10.1186/s12870-021-03143-x · Cited by 67 · see citing works
Plant Virus-Derived Vectors for Plant Genome Engineering
Mahmood, M. A., Naqvi, R. Z., Rahman, S. U., Amin, I. U., & Mansoor, S. (2023) · Viruses · doi:10.3390/v15020531 · Cited by 69 · see citing works
Novel Plant Breeding Techniques Shake Hands with Cereals to Increase Production
Haroon, M., Zafar, M. M., Farooq, M. A., Afzal, R., Batool, M., Idrees, F., Babar, U., Khan, A. S., 默辉娟, Li, L., et al. (2022) · Plants · doi:10.3390/plants11081052 · Cited by 70 · see citing works
Távora, F. T. P. K., Diniz, F. D. A. D. S., Rêgo-Machado, C. M., Freitas, N. C., Arraes, F. B. M., de Andrade, E. C., Furtado, L. L., Osiro, K. O., de Sousa, N. L., Cardoso, T. B., et al. (2022) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2022.913728 · Cited by 72 · see citing works
Genome Editing in Agriculture: Technical and Practical Considerations
Jansing, J., Schiermeyer, A., Schillberg, S., Fischer, R., & Bortesi, L. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20122888 · Cited by 66 · see citing works
Nanoparticles for protein delivery in planta
Wang, J. W.-T., Cunningham, F. J., Goh, N. S., Boozarpour, N. N., Pham, M., & Landry, M. P. (2021) · Current Opinion in Plant Biology · doi:10.1016/j.pbi.2021.102052 · Cited by 64 · see citing works
Collonnier, C., Guyon‐Debast, A., Maclot, F., Mara, K., Charlot, F., & Nogué, F. (2017) · Methods · doi:10.1016/j.ymeth.2017.04.024 · Cited by 62 · see citing works
Genome editing reagent delivery in plants
Ghogare, R., Ludwig, Y., Bueno, G. M., Slamet-Loedin, I. H., & Dhingra, A. (2021) · Transgenic Research · doi:10.1007/s11248-021-00239-w · Cited by 61 · see citing works
Kumari, C., Sharma, M., Kumar, V., Sharma, R., Kumar, V., Sharma, P., Kumar, P., & Irfan, M. (2022) · Bioengineering · doi:10.3390/bioengineering9040176 · Cited by 60 · see citing works
Luo, M., Gilbert, B., & Ayliffe, M. (2016) · Plant Cell Reports · doi:10.1007/s00299-016-1989-8 · Cited by 60 · see citing works
Lee, M. H., Lee, J., Choi, S. A., Kim, Y., Koo, O. J., Choi, S. H., Ahn, W. S., Jie, E. Y., & Kim, S. W. (2020) · Plant Biotechnology Reports · doi:10.1007/s11816-020-00645-2 · Cited by 59 · see citing works
CRISPR/Cas9-mediated genome editing in plants
Liu, X., Xie, C., Si, H., & Yang, J. (2017) · Methods · doi:10.1016/j.ymeth.2017.03.009 · Cited by 58 · see citing works
Genome engineering in ornamental plants: Current status and future prospects
Kishi‐Kaboshi, M., Aida, R., & Sasaki, K. (2018) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2018.03.015 · Cited by 59 · see citing works
Genes and genome editing tools for breeding desirable phenotypes in ornamentals
Giovannini, A., Laura, M., Nesi, B., Savona, M. R., & Cardi, T. (2021) · Plant Cell Reports · doi:10.1007/s00299-020-02632-x · Cited by 55 · see citing works
Farinati, S., Draga, S., Betto, A., Palumbo, F., Vannozzi, A., Lucchin, M., & Barcaccia, G. (2023) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1223861 · Cited by 54 · see citing works
Heritability of targeted gene modifications induced by plant-optimized CRISPR systems
Mao, Y., Botella, J. R., & Zhu, J. (2016) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-016-2380-1 · Cited by 55 · see citing works
Non-GM Genome Editing Approaches in Crops
Gong, Z., Cheng, M., & Botella, J. R. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.817279 · Cited by 51 · see citing works
Bertini, E., Tornielli, G. B., Pezzotti, M., & Zenoni, S. (2019) · Plant Cell Tissue and Organ Culture (PCTOC) · doi:10.1007/s11240-019-01619-1 · Cited by 53 · see citing works
DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants
Park, J., & Choe, S. (2019) · Transgenic Research · doi:10.1007/s11248-019-00136-3 · Cited by 51 · see citing works
Naing, A. H., Adedeji, O. S., & Kim, C. K. (2021) · Scientia Horticulturae · doi:10.1016/j.scienta.2021.110043 · Cited by 51 · see citing works
Concerns regarding ‘off-target’activity of genome editing endonucleases
Kadam, U. S., Shelake, R. M., Chavhan, R. L., & Suprasanna, P. (2018) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2018.03.027 · Cited by 47 · see citing works
Ali, Q., Yu, C., Hussain, A., Ali, M., Ahmar, S., Sohail, M. A., Riaz, M., Ashraf, M. F., Abdalmegeed, D., Wang, X., et al. (2022) · Frontiers in Plant Science · doi:10.3389/fpls.2022.860281 · Cited by 50 · see citing works
Tariq, A., Mushtaq, M., Yaqoob, H., Bhat, B. A., Zargar, S. M., Raza, A., Ali, S., Charagh, S., Mubarik, M. S., Zaman, Q. U., et al. (2023) · GM Crops & Food · doi:10.1080/21645698.2023.2219111 · Cited by 45 · see citing works
Recent advancements in CRISPR/Cas technology for accelerated crop improvement
Das, D., Singha, D. L., Paswan, R. R., Chowdhury, N., Sharma, M., Reddy, P. S., & Chikkaputtaiah, C. (2022) · Planta · doi:10.1007/s00425-022-03894-3 · Cited by 47 · see citing works
CRISPR/Cas9 and Nanotechnology Pertinence in Agricultural Crop Refinement
Naik, B. J., Shimoga, G. D., Kim, S.-C., Manjulatha, M., Palem, R. R., Palem, R. R., Kumar, M., Kim, S., & Lee, S. (2022) · Frontiers in Plant Science · doi:10.3389/fpls.2022.843575 · Cited by 44 · see citing works
Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing
Nicolia, A., Andersson, M., Hofvander, P., Festa, G., & Cardi, T. (2020) · Plant Cell Tissue and Organ Culture (PCTOC) · doi:10.1007/s11240-020-01954-8 · Cited by 44 · see citing works
Strategic transgene-free approaches of CRISPR-based genome editing in plants
Bhattacharjee, S., Bhowmick, R., Kant, L., & Paul, K. (2023) · Molecular Genetics and Genomics · doi:10.1007/s00438-023-01998-3 · Cited by 43 · see citing works
Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing
Ahn, C. H., Ramya, M., An, H. R., Park, P. M., Kim, Y.-J., Lee, S. Y., & Jang, S. (2020) · Plants · doi:10.3390/plants9060687 · Cited by 45 · see citing works
Kang, H. H., Naing, A. H., & Kim, C. K. (2020) · Biology · doi:10.3390/biology9080228 · Cited by 43 · see citing works
Delivery of CRISPR/Cas Components into Higher Plant Cells for Genome Editing
Кулуев, Б. Р., Гумерова, Г. Р., Mikhaylova, E. V., Геращенков, Г. А., Rozhnova, N. A., Vershinina, Z. R., Khyazev, A. V., Matniyazov, R. T., Baymiev, A. K., Baymiev, A. K., et al. (2019) · Russian Journal of Plant Physiology · doi:10.1134/s102144371905011x · Cited by 41 · see citing works
Bhattacharyya, N., Anand, U., Kumar, R. S., Ghorai, M., Aftab, T., Jha, N. K., Rajapaksha, A. U., Bundschuh, J., Bontempi, E., & Dey, A. (2022) · Environmental Chemistry Letters · doi:10.1007/s10311-022-01474-1 · Cited by 39 · see citing works
CRISPR/Cas genome editing in plants: mechanisms, applications, and overcoming bottlenecks
Hwarari, D., Radani, Y., Ke, Y., Chen, J., & Yang, L. (2024) · Functional & Integrative Genomics · doi:10.1007/s10142-024-01314-1 · Cited by 39 · see citing works
Application and development of genome editing technologies to the Solanaceae plants
Yamamoto, T., Kashojiya, S., Kamimura, S., Kameyama, T., Ariizumi, T., Ezura, H., & Miura, K. (2018) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2018.02.019 · Cited by 40 · see citing works
Plant biomacromolecule delivery methods in the 21st century
Rustgi, S., Naveed, S., Windham, J., Zhang, H., & Demirer, G. S. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2022.1011934 · Cited by 37 · see citing works
Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat
Liang, Z., Chen, K., & Gao, C. (2019) · Methods in molecular biology · doi:10.1007/978-1-4939-8991-1_24 · Cited by 36 · see citing works
Plant-based engineering for production of high-valued natural products
Sirirungruang, S., Markel, K., & Shih, P. M. (2022) · Natural Product Reports · doi:10.1039/d2np00017b · Cited by 34 · see citing works
Raising Climate-Resilient Crops: Journey From the Conventional Breeding to New Breeding Approaches
Gaba, Y., Pareek, A., & Singla-Pareek, S. L. (2021) · Current Genomics · doi:10.2174/1389202922666210928151247 · Cited by 35 · see citing works
Sharma, P., Pandey, A., Malviya, R., Dey, S., Karmakar, S., & Gayen, D. (2023) · Frontiers in Genome Editing · doi:10.3389/fgeed.2023.1094965 · Cited by 36 · see citing works
Banakar, R., Schubert, M. S., Kurgan, G., Mohan, K., Beaudoin, S. F., Collingwood, M. A., Vakulskas, C. A., Wang, K., & Zhang, F. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.760820 · Cited by 32 · see citing works
Targeted genome editing in polyploids: lessons from Brassica
Ahmad, N., Fatima, S., Mehmood, M. A., Zaman, Q. U., Atif, R. M., Zhou, W., Rahman, M., & Gill, R. A. (2023) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1152468 · Cited by 31 · see citing works
Inducible CRISPR genome-editing tool: classifications and future trends
Dai, X., Chen, X., Fang, Q., Li, J., & Bai, Z. (2017) · Critical Reviews in Biotechnology · doi:10.1080/07388551.2017.1378999 · Cited by 31 · see citing works
Subburaj, S., & Agapito-Tenfen, S. Z. (2023) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1255819 · Cited by 29 · see citing works
Self-citationGenome Editing as A Versatile Tool to Improve Horticultural Crop Qualities
Chen, Y., Mao, W., Liu, T., Feng, Q., Li, L., & Li, B. (2020) · Horticultural Plant Journal · doi:10.1016/j.hpj.2020.11.004 · Cited by 29 · see citing works
Levengood, H., Zhou, Y., & Zhang, C. (2024) · Plant Cell Reports · doi:10.1007/s00299-024-03359-9 · Cited by 27 · see citing works
Bai, L., Cheng, Y., She, J., He, Z., Liu, H., Zhang, G., Cao, R., & Chen, Y. (2020) · Plant Cell Tissue and Organ Culture (PCTOC) · doi:10.1007/s11240-020-01932-0 · Cited by 27 · see citing works
Genome Editing: Revolutionizing the Crop Improvement
Saurabh, S. (2021) · Plant Molecular Biology Reporter · doi:10.1007/s11105-021-01286-7 · Cited by 27 · see citing works
- Authors
- Subburaj, S., Chung, S. J., Lee, C., Ryu, S.-M., Kim, D. H., Kim, J.-S., Bae, S., & Lee, G.-J.
- Affiliations
- Chungnam National University (KR)
- Seoul National University (KR)
- Hanyang University (KR)
- Corresponding author
- Bae, Sangsu
- Lee, Geung-Joo
- Venue
- Plant Cell Reports
- Published
- 2016-01-29
- Volume
- 35
- Issue
- 7
- Pages
- 1535-1544
- ISSN
- 0721-7714 (ISSN-L)
- 1432-203X
- OpenAlex
- W2469880247
- Cited by
- 237
- References
- 55
- Citations by year
- 2026: 11 · 2025: 12 · 2024: 23 · 2023: 28 · 2022: 35 · 2021: 35 · 2020: 29 · 2019: 25 · 2018: 13 · 2017: 14 · 2016: 12
- FWCI
- 11.09
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Plant Cell Reports (publishedVersion)
- PubMed (publishedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Transgenic Plants and Applications
- Keywords
- CRISPR/Cas9, site-directed mutagenesis, Petunia hybrida, protoplast transformation, nitrate reductase, single guide RNA, ribonucleoprotein delivery, mutation efficiency, plant genome editing
- MeSH terms
- Base Sequence, Genetic Engineering, Microscopy, Fluorescence, Models, Genetic, Plant Proteins, Protoplasts, Recombinant Proteins, Ribonucleoproteins, Sequence Homology, Nucleic Acid, Mutagenesis, Site-Directed, Genome, Plant, Petunia, High-Throughput Nucleotide Sequencing, CRISPR-Cas Systems
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