DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration

Abstract

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. As proof of concept, a single-copy green fluorescent protein reporter gene (GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. The regeneration of GFP - protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.

Acknowledgements

The authors would like to thank Dr. Chiara Foresti for her kind support with the western blots and fluorescence assays. This research was funded by the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant no. 754345 awarded to SN, the University of Verona in the framework of the Grant Ricerca di Base “Definition of master regulator genes of fruit ripening in grapevine” awarded to SZ, and the Ministero delle Politiche Agricole Alimentari e Forestali (Mipaaf) in the framework of the BIOTECH-VITECH (CIG: 8704614AB4) project awarded to SZ.

Author contributions

S.N., E.B. and S.Z. designed the research; S.N. performed the research and analyzed the data; E.B contributed to data analysis; E.D., M.F., S.N. and S.Z. wrote the paper.

Data availability

The authors confirm that all the experimental data are available and accessible via the main text and/or the supplementary information.

Conflict of interests

The authors declare no conflict of interest.

References

[1] Bae S, Kweon J, Kim HS et al. Microhomology-based choice of Cas9 nuclease target sites. Nat Methods. 2014; 11: 705-6.

[2] Baltes NJ, Gil-Humanes J, Cermak T et al. DNA replicons for plant genome engineering. Plant Cell. 2014; 26: 151-63.

[3] Bertini E, Tornielli GB, Pezzotti M et al. Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars. Plant Cell, Tissue and Organ Culture (PCTOC). 2019; 138: 239-46.

[4] Clemens M, Faralli M, Lagreze J et al. VvEPFL9-1 Knock-out via CRISPR/Cas9 reduces Stomatal density in grapevine. Front Plant Sci. 2022; 13: 1-17; 878001.

[5] Concordet JP, Haeussler M . CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018; 46: W242-5.

[6] Dalla Costa L, Piazza S, Campa M et al. Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine. Plant Cell, Tissue and Organ Culture (PCTOC). 2015; 124: 471-81.

[7] Dalla Costa L, Piazza S, Pompili V et al. Strategies to produce T-DNA free CRISPRed fruit trees via agrobacterium tumefaciens stable gene transfer. Sci Rep. 2020; 10: 20155.

[8] Dhekney SA, Li ZT, Compton ME et al. Optimizing initiation and maintenance of Vitis Embryogenic cultures. Hort Science. 2009; 44: 1400-6.

[9] Driver JA, Kuniyuki AH . In vitro propagation of paradox walnut rootstock. Hort Science. 1984; 19: 507-9.

[10] Gribaudo I, Gambino G, Boccacci P et al. A multi-year study on the regenerative potential of several Vitis genotypes. Acta Hortic. 2017; 1155: 45-50.

[11] Iocco P, Franks T, Thomas MR . Genetic transformation of major wine grape cultivars of Vitis vinifera L. Transgenic Res. 2001; 10: 105-12.

[12] Iocco-Corena P, Chaïb J, Torregrosa L et al. VviPLATZ1 is a major factor that controls female flower morphology determination in grapevine. Nat Commun. 2021; 12: 6995.

[13] Jinek M, Chylinski K, Fonfara I et al. A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337: 816-21.

[14] Kanchiswamy CN . DNA-free genome editing methods for targeted crop improvement. Plant Cell Rep. 2016; 35: 1469-74.

[15] Li MY, Jiao YT, Wang YT et al. CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.). Hortic Res. 2020; 7: 149.

[16] Li ZT, Dhekney SA, Dutt M et al. An improved protocol for agrobacterium-mediated transformation of grapevine (Vitis vinifera L.). Plant Cell Tissue Organ Cult. 2008; 93: 311-21.

[17] Li ZT, Kim KH, Dhekney SA et al. An optimized procedure for plant recovery from somatic embryos significantly facilitates the genetic improvement of Vitis. Hortic Res. 2014; 1: 14027.

[18] Liang Z, Chen K, Li T et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun. 2017; 8: 14261.

[19] Liu J, Gaj T, Yang Y et al. Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nat Protoc. 2015; 10: 1842-59.

[20] Ma X, Zhu Q, Chen Y et al. CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant. 2016; 9: 961-74.

[21] Malnoy M, Viola R, Jung MH et al. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 Ribonucleoproteins. Front Plant Sci. 2016; 7: 1-9.

[22] Martinelli L, Gribaudo I . Somatic Embryogenesis in Grapevine. In: Roubelakis-Angelakis KA, ed. Molecular Biology and Biotechnology of the Grapevine. Kluwer Academic Publishers: Dordrecht, NL, 2001, 327-51.

[23] Martinez-Esteso MJ, Vilella-Anton MT, Pedreno MA et al. iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening. BMC Plant Biol. 2013; 13: 1-20.

[24] Massel K, Lam Y, Wong ACS et al. Hotter, drier, CRISPR: the latest edit on climate change. Theor Appl Genet. 2021; 134: 1691-709.

[25] Nakajima I, Ban Y, Azuma A et al. CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS One. 2017; 12: 1-16.

[26] Nishitani C, Hirai N, Komori S et al. Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep. 2016; 6: 31481.

[27] Osakabe Y, Liang Z, Ren C et al. CRISPR-Cas9-mediated genome editing in apple and grapevine. Nat Protoc. 2018; 13: 2844-63.

[28] Pan C, Ye L, Qin L et al. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep. 2016; 6: 24765.

[29] Reed KM, Bargmann BOR . Protoplast regeneration and its use in new plant breeding technologies. Front Genome Ed. 2021; 3: 1-26.

[30] Ren C, Guo Y, Kong J et al. Knockout of VvCCD8 gene in grapevine affects shoot branching. BMC Plant Biol. 2020; 20: 47.

[31] Ren C, Liu X, Zhang Z et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in chardonnay (Vitis vinifera L.). Sci Rep. 2016; 6: 32289.

[32] Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013; 162: 1618-31.

[33] Scintilla, S., Salvagnin, U., Giacomelli, L., et al. (2021) Regeneration of Plants from DNA-Free Edited Grapevine Protoplasts. https://doi.org/10.1101/2021.07.16.452503.

[34] Subburaj S, Chung SJ, Lee C et al. Site-directed mutagenesis in petunia x hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep. 2016; 35: 1535-44.

[35] Sunitha S, Rock CD . CRISPR/Cas9-mediated targeted mutagenesis of TAS4 and MYBA7 loci in grapevine rootstock 101-14. Transgenic Res. 2020; 29: 355-67.

[36] Svitashev S, Schwartz C, Lenderts B et al. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun. 2016; 7: 13274.

[37] Torregrosa L, Vialet S, Aldiveze A et al. Grapevine (V. vinifera L.). In: Wang K, ed. Agrobacterium Protocols: Volume 2. Springer Science+Business Media: New York, NY (USA), 2015, 177-94.

[38] Tu M, Fang J, Zhao R et al. CRISPR/Cas9-mediated mutagenesis of VvbZIP36 promotes anthocyanin accumulation in grapevine (Vitis vinifera). Hortic Res. 2022; 9: uhac022.

[39] Vezzulli S, Gramaje D, Tello J et al. Genomic Designing for Biotic Stress Resistant Grapevine. In: Kole C, ed. Genomic Designing for Biotic Stress Resistant Fruit Crops. Springer: Cham, 2022, 87-255.

[40] Wan DY, Guo Y, Cheng Y et al. CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera). Hortic Res. 2020; 7: 116.

[41] Wang M, Lu Y, Botella JR et al. Gene targeting by homology-directed repair in Rice using a Geminivirus-based CRISPR/Cas9 system. Mol Plant. 2017; 10: 1007- 10.

[42] Wang X, Tu M, Wang D et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant Biotechnol J. 2018; 16: 844-55.

[43] Webb LB, Whetton PH, Barlow EWR . Modelled impact of future climate change on the phenology of winegrapes in Australia. Aust J Grape Wine Res. 2007; 13: 165-75.

[44] Woo JW, Kim J, Kwon SI et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol. 2015; 33: 1162-4.

[45] Xie K, Yang Y . RNA_guided genome editibg in plant using a CRSPR-Cas system. Mol Plant. 2013; 6: 1975-83.

[46] Xing HL, Dong L, Wang ZP et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014; 14: 327.

[47] Zhang Y, Liang Z, Zong Y et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun. 2016; 7: 12617.

[48] Zhang Y, Massel K, Godwin ID et al. Applications and potential of genome editing in crop improvement. Genome Biol. 2018; 19: 210.

[49] Zhu YM, Hoshino Y, Nakano M et al. Highly efficient system of plant regeneration from protoplasts of grapevine (Vitis vinzfera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. Plant Sci. 1997; 123: 151-7.