Abstract
Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aiken GR, Hsu-Kim H, Ryan JN (2011) Influence of dissolved organic matter on the environmental fate of metals, nanoparticles, and colloids
Ali Z, Abul-Faraj A, Li L et al (2015) Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant 8:1288–1291
Almofti MR, Harashima H, Shinohara Y et al (2003) Lipoplex size determines lipofection efficiency with or without serum. Mol Membr Biol 20:35–43
Alsaiari SK, Patil S, Alyami M et al (2018) Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. J Am Chem Soc 140:143–146
Andersson M, Turesson H, Olsson N et al (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant 164:378–384. https://doi.org/10.1111/ppl.12731
Baek K, Kim DH, Jeong J et al (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6:30620
Baltes NJ, Gil-Humanes J, Voytas DF (2017) Chapter One - Genome Engineering and Agriculture: Opportunities and Challenges. In: Weeks DP, Yang BBT-P in MB and TS (eds) Gene Editing in Plants. Academic Press, pp 1–26
Banakar R, Eggenberger AL, Lee K et al (2019) High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice. Sci Rep 9:19902. https://doi.org/10.1038/s41598-019-55681-y
Baysal C, Bortesi L, Zhu C et al (2016) CRISPR/Cas9 activity in the rice OsBEIIb gene does not induce off-target effects in the closely related paralog OsBEIIa. Mol Breed 36:108. https://doi.org/10.1007/s11032-016-0533-4
Bevan MW, Flavell RB, Chilton M-D (1984) A chimeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184–187
Bhowmik P, Ellison E, Polley B et al (2018) Targeted mutagenesis in wheat microspores using CRISPR/Cas9. Sci Rep 8:6502. https://doi.org/10.1038/s41598-018-24690-8
De BM, Herrera-Estrella L, Van MM et al (1984) Expression of foreign genes in regenerated plants and their progeny. EMBO J 3:1681–1689
Carbone S, Gatti A, Vianello G, Nannipieri P (2014) Uptake and translocation of metals and nutrients in tomato grown in soil polluted with metal oxide (CeO2, Fe3O2, SnO2, TiO2) or metallic (Ag Co, Ni) engineered nanoparticles. Environ Sci Pollut Res Int 22:1841–1853
Chang F-P, Kuang L-Y, Huang C-A et al (2013) A simple plant gene delivery system using mesoporous silica nanoparticles as carriers. J Mater Chem B 1:5279–5287
Chaverra-Rodriguez D, Macias VM, Hughes GL et al (2018) Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat Commun 9:1–11
Chen K, Wang Y, Zhang R et al (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol 70:667–697. https://doi.org/10.1146/annurev-arplant-050718-100049
Chen L, Li W, Katin-Grazzini L et al (2018) A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants. Hortic Res 5:1–12
Christou P (1993) Particle gun mediated transformation. Curr Opin Biotechnol 4:135–141. https://doi.org/10.1016/0958-1669(93)90113-B
Cifuentes Z, Custardoy L, de la Fuente JM et al (2010) Absorption and translocation to the aerial part of magnetic carbon-coated nanoparticles through the root of different crop plants. J Nanobiotechnology 8:1–8
Clark DP, Pazdernik NJ (2016) Chapter 15 - Transgenic Plants and Plant Biotechnology. In: Second E (ed) Clark DP, Pazdernik NJBT-B. Academic Cell, Boston, pp 461–492
Cody WB, Scholthof HB (2019) Plant virus vectors 3.0: Transitioning into synthetic genomics. Annu Rev Phytopathol 57:211–230
Cong L, Ran FA, Cox D, et al (2013) Multiplex genome engineering using CRISPR/Cas SYSTEMS. Science (80- ) 339:819 LP – 823. doi: https://doi.org/10.1126/science.1231143
Corredor E, Testillano PS, Coronado M-J et al (2009) Nanoparticle penetration and transport in living pumpkin plants: in situ subcellular identification. BMC Plant Biol 9:1–11
Cunningham FJ, Goh NS, Demirer GS et al (2018) Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol 36:882–897
Daya S, Berns KI (2008) Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21:583–593
Demirer GS, Landry MP (2017) Delivering genes to plants. In: Chemical engineering progress. pp 40–45
Demirer GS, Zhang H, Goh NS et al (2019a) Carbon nanotube–mediated DNA delivery without transgene integration in intact plants. Nat Protoc 14:2954–2971
Demirer GS, Zhang H, Matos JL et al (2019b) High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat Nanotechnol 14:456–464
Fabrega J, Fawcett SR, Renshaw JC, Lead JR (2009) Silver nanoparticle impact on bacterial growth: effect of pH, concentration, and organic matter. Environ Sci Technol 43:7285–7290
Feng Z, Zhang B, Ding W et al (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. https://doi.org/10.1038/cr.2013.114
Finke S, Conzelmann K-K (2005) Recombinant rhabdoviruses: vectors for vaccine development and gene therapy. In: The World of Rhabdoviruses. Springer, pp 165–200
Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405. https://doi.org/10.1016/j.tibtech.2013.04.004
Gallois J-L, Woodward C, Reddy GV, Sablowski R (2002) Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis. Development 129:3207–3217
Gerhardt R (2011) Properties and applications of silicon carbide. BoD–Books on Demand
Ghogare R, Williamson-Benavides B, Ramírez-Torres F, Dhingra A (2019) CRISPR-associated nucleases: the Dawn of a new age of efficient crop improvement. Transgenic Res 1–35
Glass Z, Lee M, Li Y, Xu Q (2018) Engineering the Delivery System for CRISPR-Based Genome Editing. Trends Biotechnol 36:173–185. https://doi.org/10.1016/j.tibtech.2017.11.006
Guo B, Itami J, Oikawa K et al (2019) Native protein delivery into rice callus using ionic complexes of protein and cell-penetrating peptides. PLoS ONE 14:e0214033–e0214033. https://doi.org/10.1371/journal.pone.0214033
Hajiahmadi Z, Shirzadian-Khorramabad R, Kazemzad M, Sohani MM (2019) Enhancement of tomato resistance to Tuta absoluta using a new efficient mesoporous silica nanoparticle-mediated plant transient gene expression approach. Sci Hortic (Amsterdam) 243:367–375. https://doi.org/10.1016/j.scienta.2018.08.040
Hamada H, Liu Y, Nagira Y et al (2018) Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat. Sci Rep 8:14422. https://doi.org/10.1038/s41598-018-32714-6
Hamilton CM, Frary A, Lewis C, Tanksley SD (1996) Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci U S A 93:9975–9979. https://doi.org/10.1073/pnas.93.18.9975
Han G, Ghosh P, Rotello VM (2007) Functionalized gold nanoparticles for drug delivery. Nanomedicine 2:113–123. https://doi.org/10.2217/17435889.2.1.113
Hao Y, Yang X, Shi Y et al (2013) Magnetic gold nanoparticles as a vehicle for fluorescein isothiocyanate and DNA delivery into plant cells. Botany 91:457–466. https://doi.org/10.1139/cjb-2012-0281
Hu J, Li S, Li Z et al (2019) A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize. Mol Plant Pathol 20:1463–1474
Jansing J, Schiermeyer A, Schillberg S et al (2019) Genome editing in agriculture: Technical and practical considerations. Int J Mol Sci 20:2888. https://doi.org/10.3390/ijms20122888
Jiang L, Ding L, He B et al (2014) Systemic gene silencing in plants triggered by fluorescent nanoparticle-delivered double-stranded RNA. Nanoscale 6:9965–9969
Jiang W, Zhou H, Bi H et al (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. https://doi.org/10.1093/nar/gkt780
Kaya H, Ishibashi K, Toki S (2017) A split Staphylococcus aureus Cas9 as a compact genome-editing tool in plants. Plant Cell Physiol 58(4):643–649
Kim H, Kim S-T, Ryu J et al (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8:14406
Kwak S-Y, Lew TTS, Sweeney CJ et al (2019) Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat Nanotechnol 14:447–455. https://doi.org/10.1038/s41565-019-0375-4
Lange M, Yellina AL, Orashakova S, Becker A (2013) Virus-induced gene silencing (VIGS) in plants: an overview of target species and the virus-derived vector systems. In: Virus-Induced Gene Silencing. Springer, pp 1–14
Lee B, Lee K, Panda S et al (2018) Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat Biomed Eng 2:497–507
Li JF, Norville JE, Aach J et al (2013) Multiplex and homologous recombination-mediated genome editing in arabidopsis and nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. https://doi.org/10.1038/nbt.2654
Li Z, Liu Z-B, Xing A, et al (2015) Cas9-Guide RNA Directed Genome Editing in Soybean. Plant Physiol 169:960 LP – 970. doi: https://doi.org/10.1104/pp.15.00783
Liang X, Potter J, Kumar S et al (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53
Liang Z, Chen K, Li T et al (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261
Liang Z, Chen K, Zhang Y et al (2018) Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat Protoc 13:413–430. https://doi.org/10.1038/nprot.2017.145
Liang Z, Zhang K, Chen K, Gao C (2014) Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System. J Genet Genomics 41:63–68. https://doi.org/10.1016/j.jgg.2013.12.001
Lino CA, Harper JC, Carney JP, Timlin JA (2018) Delivering CRISPR: a review of the challenges and approaches. Drug Deliv 25:1234–1257. https://doi.org/10.1080/10717544.2018.1474964
Liu W, Rudis MR, Cheplick MH et al (2020) Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Rep 39:245–257. https://doi.org/10.1007/s00299-019-02488-w
Lowe K, La Rota M, Hoerster G et al (2018) Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis. Vitr Cell Dev Biol 54:240–252
Lowe K, Wu E, Wang N et al (2016) Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28:1998–2015
Ma X, Zhang X, Liu H, Li Z (2020) Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9. Nat Plants 6:773–779
Maher MF, Nasti RA, Vollbrecht M et al (2020) Plant gene editing through de novo induction of meristems. Nat Biotechnol 38:84–89. https://doi.org/10.1038/s41587-019-0337-2
Malnoy M, Viola R, Jung MH et al (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7:1–9. https://doi.org/10.3389/fpls.2016.01904
Martin-Ortigosa S, Valenstein JS, Lin VS-Y et al (2012) Gold functionalized mesoporous silica nanoparticle mediated protein and DNA Codelivery to plant cells via the biolistic method. Adv Funct Mater 22:3576–3582. https://doi.org/10.1002/adfm.201200359
Matsumoto TK, Gonsalves D (2012) 8 - Biolistic and other non-Agrobacterium technologies of plant transformation. In: Altman A, Hasegawa PMBT-PB and A (eds). Academic Press, San Diego, pp 117–129
Miao J, Guo D, Zhang J et al (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23:1233–1236. https://doi.org/10.1038/cr.2013.123
Mitter N, Worrall EA, Robinson KE et al (2017) Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat plants 3:1–10
Murovec J, Guček K, Bohanec B, et al (2018) DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes . Front. Plant Sci. 9:1594
Nekrasov V, Staskawicz B, Weigel D et al (2013) Targeted mutagenesis in the model plant nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. https://doi.org/10.1038/nbt.2655
Nelson-Vasilchik K, Hague J, Mookkan M et al (2018) Transformation of recalcitrant sorghum varieties facilitated by baby boom and Wuschel2. Curr Protoc Plant Biol 3:e20076
Onelli E, Prescianotto-Baschong C, Caccianiga M, Moscatelli A (2008) Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold. J Exp Bot 59:3051–3068
Osakabe Y, Liang Z, Ren C et al (2018) CRISPR–Cas9-mediated genome editing in apple and grapevine. Nat Protoc 13:2844–2863
Ozyigit II (2020) Gene transfer to plants by electroporation: methods and applications. Mol Biol Rep 47:3195–3210. https://doi.org/10.1007/s11033-020-05343-4
Peretyazhko TS, Zhang Q, Colvin VL (2014) Size-controlled dissolution of silver nanoparticles at neutral and acidic pH conditions: kinetics and size changes. Environ Sci Technol 48:11954–11961
Phenrat T, Cihan A, Kim H-J et al (2010) Transport and deposition of polymer-modified Fe0 nanoparticles in 2-D heterogeneous porous media: Effects of particle concentration, Fe0 content, and coatings. Environ Sci Technol 44:9086–9093
Raliya R, Franke C, Chavalmane S et al (2016a) Quantitative understanding of nanoparticle uptake in watermelon plants. Front Plant Sci 7:1288
Raliya R, Tarafdar JC, Biswas P (2016b) Enhancing the mobilization of native phosphorus in the mung bean rhizosphere using zno nanoparticles synthesized by soil fungi. J Agric Food Chem 64:3111–3118. https://doi.org/10.1021/acs.jafc.5b05224
Ramakrishna S, Dad A-BK, Beloor J et al (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–1027
Ran FA, Cong L, Yan WX et al (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186
Razzaq A, Saleem F, Kanwal M et al (2019) Modern trends in plant genome editing: an inclusive review of the CRISPR/Cas9 toolbox. Int J Mol Sci 20:4045
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355. https://doi.org/10.1038/nbt.2842
Sandhya D, Jogam P, Allini VR et al (2020) The present and potential future methods for delivering CRISPR/Cas9 components in plants. J Genet Eng Biotechnol 18:25. https://doi.org/10.1186/s43141-020-00036-8
Schumann K, Lin S, Boyer E et al (2015) Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci 112:10437–10442
Shan Q, Wang Y, Li J et al (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. https://doi.org/10.1038/nbt.2650
Shi J, Gao H, Wang H et al (2017) ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol J 15:207–216. https://doi.org/10.1111/pbi.12603
Su Y, Ashworth V, Kim C et al (2019) Delivery, uptake, fate, and transport of engineered nanoparticles in plants: a critical review and data analysis. Environ Sci Nano 6:2311–2331
Su Y, Qian D, Adeleye AS et al (2018) Impact of ageing on the fate of molybdate-zerovalent iron nanohybrid and its subsequent effect on cyanobacteria (Microcystis aeruginosa) growth in aqueous media. Water Res 140:135–147
Subburaj S, Chung SJ, Lee C et al (2016) Site-directed mutagenesis in Petunia× hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35:1535–1544
Svitashev S, Schwartz C, Lenderts B et al (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7:13274
Torney F, Trewyn BG, Lin VS-Y, Wang K (2007) Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2:295–300
Vijayakumar PS, Abhilash OU, Khan BM, Prasad BLV (2010) Nanogold-Loaded sharp-edged carbon bullets as plant-gene carriers. Adv Funct Mater 20:2416–2423
Wang Y, Cheng X, Shan Q et al (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947
Wolt JD, Wang K, Sashital D, Lawrence-Dill CJ (2016) Achieving plant CRISPR targeting that limits off-target effects. Plant Genome 9:1–8. https://doi.org/10.3835/plantgenome2016.05.0047
Woo JW, Kim J, Il KS et al (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164
Xie K, Yang Y (2013) RNA-Guided genome editing in plants using a CRISPR–Cas system. Mol Plant 6:1975–1983. https://doi.org/10.1093/mp/sst119
Zhai G, Walters KS, Peate DW et al (2014) Transport of gold nanoparticles through plasmodesmata and precipitation of gold ions in woody poplar. Environ Sci Technol Lett 1:146–151
Zhang H, Demirer GS, Zhang H et al (2019) DNA nanostructures coordinate gene silencing in mature plants. Proc Natl Acad Sci 116:7543–7548
Zhang H, Zhang J, Wei P et al (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807. https://doi.org/10.1111/pbi.12200
Zhang Y, Liang Z, Zong Y et al (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:1–8
Zhang Z, He X, Zhang H et al (2011) Uptake and distribution of ceria nanoparticles in cucumber plants. Metallomics 3:816–822
Zhao X, Liu W, Cai Z et al (2016) An overview of preparation and applications of stabilized zero-valent iron nanoparticles for soil and groundwater remediation. Water Res 100:245–266
Zhao X, Meng Z, Wang Y et al (2017) Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers. Nat Plants 3:956–964. https://doi.org/10.1038/s41477-017-0063-z
Zhu Z-J, Wang H, Yan B et al (2012) Effect of surface charge on the uptake and distribution of gold nanoparticles in four plant species. Environ Sci Technol 46:12391–12398
Acknowledgements
Work in the Dhingra lab in crop improvement is supported in part by Washington State University Agriculture Research Center Hatch grant WNP00011.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Ghogare, R., Ludwig, Y., Bueno, G.M. et al. Genome editing reagent delivery in plants. Transgenic Res 30, 321–335 (2021). https://doi.org/10.1007/s11248-021-00239-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-021-00239-w