Abstract
Genome editing has revolutionized genetics and breeding likewise. Especially in plant breeding, it opened new ways to address traits with never known specificity. In many cases genome editing tools are provided by classical transgenic methods, i.e., by Agrobacterium-based delivery, but it is also possible to perform genome editing without the use of a transgene by providing proteins, or nucleic acid protein complexes. These methods have the big advantage that transgene organisms can be avoided at any time; even transgenic intermediates are not needed. However, transgene-free methods are technically challenging, and editing rates are often lower compared to classical methods. Nevertheless, it offers great opportunities to produce plants without the need of any transgene, simplifying the regulatory processes in many jurisdictions around the globe. In this chapter, we present methods and delivery methods that can be used for transgene-free editing and present first promising examples.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ali Z, Mahas A, Mahfouz M (2018) CRISPR/Cas13 as a Tool for RNA Interference. Trends Plant Sci 23:374–378. Available: http://dx.doi.org/10.1016/j.tplants.2018.03.003
Aman R, Ali Z, Butt H, Mahas A, Aljedaani F, Khan MZ et al (2018) RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol 19(1):1–9
Ariga H, Toki S, Ishibashi K (2020) Potato virus X Vector-mediated DNA-Free genome editing in plants. Plant Cell Physiol 61(11):1946–1953
Augustine SM, Cherian AV, Seiling K, Di Fiore S, Raven N, Commandeur U, Schillberg S (2021) Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance. Physiol Plant 173(3):993–1007. https://doi.org/10.1111/ppl.13499. Epub 2021 Jul 28. PMID: 34265107
Badhan S, Ball AS, Mantri N (2021) First report of CRISPR/Cas9 Mediated DNA-Free Editing of 4CL and RVE7 genes in Chickpea protoplasts. Int J Mol Sci 22(1). https://doi.org/10.3390/ijms22010396
Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF (2014) DNA replicons for plant genome engineering. Plant Cell 26(1):151–163
Banakar R, Eggenberger AL, Lee K, Wright DA, Murugan K, Zarecor S et al (2019) High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice. Sci Rep 9(1):19902. https://doi.org/10.1038/s41598-019-55681-y
Banakar R, Schubert M, Collingwood M, Vakulskas C, Eggenberger AL, Wang K (2020) Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein complexes for genome editing efficiency in the Rice Phytoene Desaturase (OsPDS) gene. Rice (New York, N.Y.) 13(1):4. https://doi.org/10.1186/s12284-019-0365-z
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712
Becker S, Boch J (2016) TALEs spin along, but not around. Nature Chem Biol 12(10):766–768
Becker S, Boch J (2021) TALE and TALEN genome editing technologies. Gene Genome Editin 2:100007
Beumer KJ, Trautman JK, Christian M, Dahlem TJ, Lake CM, Hawley RS et al (2013) Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila. G3 (Bethesda, Md.) 3(10):1717–1725. https://doi.org/10.1534/g3.113.007260
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S et al (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326(5959):1509–1512
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151(8):2551–2561
Butler NM, Baltes NJ, Voytas DF, Douches DS (2016) Geminivirus-mediated genome editing in potato (Solanum tuberosum L.) using sequence-specific nucleases. Front Plant Sci 7:1045
Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C et al (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39(12):e82–e82
Cody WB, Scholthof HB, Mirkov TE (2017) Multiplexed gene editing and protein overexpression using a tobacco mosaic virus viral vector. Plant Physiol 175(1):23–35
Fan Y, Xin S, Dai X, Yang X, Huang H, Hua Y (2020) Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins. Ind Crops Prod 146:112146. https://doi.org/10.1016/j.indcrop.2020.112146
Feng Z, Zhang B, Ding W, Liu X, Yang D-L, Wei P et al (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Research 23(10):1229–1232. https://doi.org/10.1038/cr.2013.114
Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551(7681):464–471. https://doi.org/10.1038/nature24644
Göhre V, Robatzek S (2008) Breaking the barriers: microbial effector molecules subvert plant immunity. Annu Rev Phytopathol 46:189–215
González MN, Massa GA, Andersson M, Turesson H, Olsson N, Fält A-S et al (2019) Reduced enzymatic browning in potato tubers by specific editing of a Polyphenol Oxidase gene via Ribonucleoprotein complexes delivery of the CRISPR/Cas9 system. Front Plant Sci 10:1649. https://doi.org/10.3389/fpls.2019.01649
Grens K (2015) There’s CRISPR in your yogurt: we’ve all been eating food enhanced by the genome-editing tool for years. Scientist 29(1):1–5
Grohmann L, Keilwagen J, Duensing N, Dagand E, Hartung F, Wilhelm R et al (2019) Detection and identification of genome editing in plants: challenges and opportunities. Front Plant Sci 10:236
Guo B, Itami J, Oikawa K, Motoda Y, Kigawa T, Numata K (2019) Native protein delivery into rice callus using ionic complexes of protein and cell-penetrating peptides. PloS One 14(7):e0214033. https://doi.org/10.1371/journal.pone.0214033
Huang T-K, Puchta H (2021) Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering. Trans Res. https://doi.org/10.1007/s11248-021-00238-x
Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169(12):5429–5433
Jansen R, van Embden J, Gaastra W, Schouls LM (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43(6):1565–1575
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Kang B-C, Yun J-Y, Kim S-T, Shin YJ, Ryu J, Choi M et al (2018) Precision genome engineering through adenine base editing in plants. Nat Plant 4(7):427–431. https://doi.org/10.1038/s41477-018-0178-x
Kang S, Jeon S, Kim S, Chang YK, Kim Y-C (2020) Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii. Sci Rep 10(1):1–11
Kazama T, Okuno M, Watari Y, Yanase S, Koizuka C, Tsuruta Y et al (2019) Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing. Nat Plant 5(7):722–730
Kim H, Choi J (2021) A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins. Plant Cell Rep 40(6):1059–1070. https://doi.org/10.1007/s00299-020-02597-x
Kim H, Kim S-T, Ryu J, Kang B-C, Kim J-S, Kim S-G (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8:14406. https://doi.org/10.1038/ncomms14406
Kim J-S, Kang B-C, Bae S-J, Lee S, Lee JS, Kim A et al (2021) Chloroplast and mitochondrial DNA editing in plants. Nat Plant 7:899–905
Lee MH, Lee J, Choi SA, Kim Y-S, Koo O, Choi SH et al (2020) Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection. Plant Biotechnol Rep 14(6):695–702. https://doi.org/10.1007/s11816-020-00645-2
Li S, Song Z, Liu C, Chen X-L, Han H (2019) Biomimetic mineralization-based CRISPR/Cas9 ribonucleoprotein nanoparticles for gene editing. ACS Appl Mater Interface 11(51):47762–47770. https://doi.org/10.1021/acsami.9b17598
Lin Q, Zhu Z, Liu G, Sun C, Lin D, Xue C et al (2021) Genome editing in plants with MAD7 nuclease. J Genet Genomic 48:444–451
Liu W, Rudis MR, Cheplick MH, Millwood RJ, Yang J-P, Ondzighi-Assoume CA et al (2020) Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Rep 39(2):245–257
Ma X, Zhang X, Liu H, Li Z (2020) Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9. Nat Plants 6(7):773–779
Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ et al (2020) Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 18(2):67–83
Makhotenko AV, Khromov AV, Snigir EA, Makarova SS, Makarov VV, Suprunova TP et al (2019) Functional analysis of Coilin in virus resistance and stress tolerance of Potato Solanum tuberosum using CRISPR-Cas9 editing. Doklady Biochem Biophys 484(1):88–91. https://doi.org/10.1134/S1607672919010241
Menz J, Modrzejewski D, Hartung F, Wilhelm R, Sprink T (2020) Genome edited crops touch the market: a view on the global development and regulatory environment. Front Plant Sci 11:586027
Metje-Sprink J, Menz J, Modrzejewski D, Sprink T (2019) DNA-free genome editing: past, present and future. Front Plant Sci 9:1957
Metje-Sprink J, Sprink T, Hartung F (2020) Genome-edited plants in the field. Curr Opn Biotechnol 61:1–6
Modrzejewski D, Hartung F, Sprink T, Krause D, Kohl C, Wilhelm R (2019) What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects: a systematic map. Environ Eviden 8(1):1–33
Modrzejewski D, Hartung F, Lehnert H, Sprink T, Kohl C, Keilwagen J, Wilhelm R (2020) Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas: a systematic review in plants. Front Plant Sci 11:1838
Mojica FJM, García-Martínez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60(2):174–182
Mok BY, de Moraes MH, Zeng J, Bosch DE, Kotrys AV, Raguram A et al (2020) A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583(7817):631–637
Murovec J, Guček K, Bohanec B, Avbelj M, Jerala R (2018) DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes. Front Plant Sci 9:1594. https://doi.org/10.3389/fpls.2018.01594
Oliva R, Ji C, Atienza-Grande G, Huguet-Tapia JC, Perez-Quintero A, Li T et al (2019) Broad-spectrum resistance to bacterial blight in rice using genome editing. Nat Biotechnol 37(11):1344–1350
Pourcel C, Salvignol G, Vergnaud G (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151(3):653–663
Schmitz DJ, Ali Z, Wang C, Aljedaani F, Hooykaas PJJ, Mahfouz M, de Pater S (2020) CRISPR/Cas9 mutagenesis by translocation of Cas9 protein into plant cells via the Agrobacterium Type IV secretion system. Front Genome Editin 2:6. https://doi.org/10.3389/fgeed.2020.00006
Sprink T, Metje J, Hartung F (2015) Plant genome editing by novel tools: TALEN and other sequence specific nucleases. Curr Opn Biotechnol 32:47–53
Toda E, Koiso N, Takebayashi A, Ichikawa M, Kiba T, Osakabe K et al (2019) An efficient DNA-and selectable-marker-free genome-editing system using zygotes in rice. Nat Plants 5(4):363–368
Wang JW, Grandio EG, Newkirk GM, Demirer GS, Butrus S, Giraldo JP, Landry MP (2019) Nanoparticle-mediated genetic engineering of plants. Mol Plant 12(8):1037–1040
Wu S, Zhu H, Liu J, Yang Q, Shao X, Bi F et al (2020) Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana. BMC Plant Biology 20(1):425. https://doi.org/10.1186/s12870-020-02609-8
Yu J, Tu L, Subburaj S, Bae S, Lee G-J (2021) Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins. Plant Cell Rep 40(6):1037–1045. https://doi.org/10.1007/s00299-020-02593-1
Zhang R, Liu J, Chai Z, Chen S, Bai Y, Zong Y et al (2019) Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing. Nat Plants 5(5):480–485. https://doi.org/10.1038/s41477-019-0405-0
Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y et al (2017) Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35(5):438–440. https://doi.org/10.1038/nbt.3811
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Nature Switzerland AG
About this chapter
Cite this chapter
Sprink, T., Hartung, F., Metje-Sprink, J. (2022). Transgene-Free Genome Editing in Plants. In: Wani, S.H., Hensel, G. (eds) Genome Editing. Springer, Cham. https://doi.org/10.1007/978-3-031-08072-2_8
Download citation
DOI: https://doi.org/10.1007/978-3-031-08072-2_8
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-031-08071-5
Online ISBN: 978-3-031-08072-2
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)