Virus induced gene editing using potyviral vectors in Cas12a expressing plants

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are revolutionizing precision genome editing and gene expression control in crop plants. While effective CRISPR-Cas applications traditionally rely on labor-intensive stable genetic transformation to deliver Cas nucleases and guide RNAs into plant cells, plant viruses have emerged as a faster and efficient alternative, a strategy known as virus-induced gene editing (VIGE). Cas12a, Class 2 Type V CRISPR nucleases, are an alternative to broadly used Cas9 for plant genome engineering. Both kind of nucleases offer precise editing, but some Cas12a unique features make them particularly well suited for VIGE. In this study, we first used a tobacco rattle virus vector to compare editing efficiency of various target sequences and CRISPR RNA (crRNA) architectures in Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a)-expressing Nicotiana benthamiana plants, evaluating results in infected tissues and seeds. Next, we developed a tobacco etch virus (genus Potyvirus)-derived vector efficiently delivering crRNAs throughout the plant. This approach enabled generation of plants with all four edited alleles in the allotetraploid N. benthamiana through in vitro regeneration from infected leaves, and to produce edited non-infected progeny, although at a very low frequency. We then demonstrated the successful application of the potyviral vector for VIGE in agronomically important crops, such as tomato or cultivated tobacco. Finally, we replicated this design using two other potyviral vectors, turnip mosaic virus, and lettuce mosaic virus. Given the conserved biological properties among potyviruses, we believe these findings are broadly applicable to the largest genus of plant RNA viruses, significantly expanding the host range of the VIGE technology.

Acknowledgements

This research was supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU; Spain) through the Agencia Estatal de Investigación (CPP2021-008712, PID2022-141438OB-I00, PID2023-146418OB-I00, PID2023-151867OB-C32; MICIU/AEI/10.13039/501100011033 and ERDF, EU) and Generalitat Valenciana through program PROMETEO (CIPROM/2022/21). A.G. and M.R.-R. are the recipients of predoctoral contracts (FPU20/05477 and FPU21/00055, respectively) from MICIU.

Author contributions

F.M. and J.-A.D. conceived the work with input from the rest of the authors. F.M., A.G., U.R., I.Q.-M., B.G.-S., C.D.P., and M.R.-R. performed the experiments. All authors analyzed the data. F.M., A.G. and J.-A.D. wrote the manuscript with input from the rest of the authors. All authors discussed and revised the manuscript.

Data availability

The data that supports the findings of this study are in Supplementary material.

Conflicts of interest statement

The authors declare no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Li B, Sun C, Li J, et al. Targeted genome-modification tools and their advanced applications in crop breeding. Nat Rev Genet. 2024; 25: 603-22.

[2] Tuncel A, Pan C, Clem JS, et al. CRISPR-Cas applications in agriculture and plant research. Nat Rev Mol Cell Biol. 2025; 26: 419-41.

[3] Laforest LC, Nadakuduti SS . Advances in delivery mechanisms of CRISPR gene-editing reagents in plants. Front Genome Ed. 2022; 4: 1-10.

[4] Steinberger AR, Voytas DF . Virus-induced gene editing free from tissue culture. Nat Plants. 2025; 11: 1241-51.

[5] Khakhar A, Voytas DF . RNA viral vectors for accelerating plant synthetic biology. Front Plant Sci. 2021; 12.

[6] Ali Z, Abul-faraj A, Li L, et al. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant. 2015; 8: 1288-91.

[7] Ellison EE, Nagalakshmi U, Gamo ME, et al. Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat Plants. 2020; 6: 620-4.

[8] Hoffmann G, Incarbone M . A resilient bunch: stem cell antiviral immunity in plants. New Phytol. 2024; 241: 1415-20.

[9] Nagalakshmi U, Meier N, Liu JY, et al. High-efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus. Plant Physiol. 2022; 189: 1241-5.

[10] Liu Q, Zhao C, Sun K, et al. Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes. Mol Plant. 2023; 16: 616-31.

[11] Yoshida T, Ishikawa M, Toki S, et al. Heritable tissue-culture-free gene editing in Nicotiana benthamiana through viral delivery of SpCas9 and sgRNA. Plant Cell Physiol. 2024; 00: 1743-50.

[12] Weiss T, Kamalu M, Shi H, et al. Viral delivery of an RNA-guided genome editor for transgene-free plant germline editing. Nat Plants. 2025; 11: 967-76.

[13] Bernabé-Orts JM, Casas-Rodrigo I, Minguet EG, et al. Assessment of Cas12a-mediated gene editing efficiency in plants. Plant Biotechnol J. 2019; 17: 1971-84.

[14] Bandyopadhyay A, Kancharla N, Javalkote VS, et al. CRISPR-Cas12a (Cpf1): a versatile tool in the plant genome editing tool box for agricultural advancement. Front Plant Sci. 2020; 11: 1-17.

[15] Paul B, Montoya G . CRISPR-Cas12a: functional overview and applications. Biom J. 2020; 43: 8-17.

[16] Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163: 759-71.

[17] Yang Y, Wang D, Lü P, et al. Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system. Mol Biol Rep. 2023; 50: 3723-38.

[18] Uranga M, Vazquez-Vilar M , Orzaez D, et al. CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors. Cris J. 2021; 4: 761-9.

[19] Wylie SJ, Adams M, Chalam C, et al. ICTV virus taxonomy profile: Potyviridae. J Gen Virol. 2017; 98: 352-4.

[20] Eid A, Mohan C, Sanchez S, et al. Multiallelic, targeted mutagenesis of magnesium Chelatase with CRISPR/Cas9 provides a rapidly scorable phenotype in highly polyploid sugarcane. Front Genome Ed. 2021; 3: 654996.

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

[22] Aragonés V, Aliaga F, Pasin F, et al. Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors. Biotechnol J. 2022; 17: 2100504.

[23] García A, Aragonés V, Gioiosa S, et al. Comparative genomics-driven design of virus-delivered short RNA inserts triggering robust gene silencing. Plant Biotechnol J. 2025; 23: 4930-2.

[24] Yang L, Machin F, Wang S, et al. Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks. Nat Biotechnol. 2023; 41: 958-67.

[25] Torres-Barceló C, Martín S, Daròs JA, et al. From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the tobacco etch potyvirus HC-pro. Genetics. 2008; 180: 1039-49.

[26] Schindele P, Puchta H . Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing. Plant Biotechnol J. 2020; 18: 1118-20.

[27] Liu P, Luk K, Shin M, et al. Enhanced Cas12a editing in mammalian cells and zebrafish. Nucleic Acids Res. 2019; 47: 4169-80.

[28] López-González S, Aragonés V, Daròs JA, et al. An infectious cDNA clone of a radish-infecting turnip mosaic virus strain. Eur J Plant Pathol. 2017; 148: 207-11.

[29] Krause-Sakate R, Redondo E, Richard-Forget F, et al. Molecular mapping of the viral determinants of systemic wilting induced by a lettuce mosaic virus (LMV) isolate in some lettuce cultivars. Virus Res. 2005; 109: 175-80.

[30] Zhang C, Liu S, Li X, et al. Virus-induced gene editing and its applications in plants. Int J Mol Sci. 2022; 23: 10202.

[31] Uranga M, Daròs JA . Tools and targets: the dual role of plant viruses in CRISPR-Cas genome editing. Plant Genome. 2023; 16: 1-25.

[32] Uranga M, Aragonés V, Selma S, et al. Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a potato virus X vector. Plant J. 2021; 106: 555-65.

[33] Sun T, Wang P, Rao S, et al. Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants. Mol Plant. 2023; 16: 1048-65.

[34] Huang YS, Li HM . Arabidopsis CHLI2 can substitute for CHLI1. Plant Physiol. 2009; 150: 636-45.

[35] Zetsche B, Heidenreich M, Mohanraju P, et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol. 2017; 35: 31-4.

[36] Oh Y, Nagalakshmi U, Dahlbeck D, et al. Heritable virus-induced germline editing in tomato. Plant J. 2025; 122: 1-9.

[37] Lee HJ, Baik JE, Kim KN . Development of an efficient and heritable virus-induced genome editing system in Solanum lycopersicum. Hortic Res. 2025; 12: 1-10.

[38] Qiao J-H, Zang Y, Gao Q, et al. Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat. Nat Plants. 2025; 11: 1252-9. https://doi.org/10.1038/s41477-025-02023-8.

[39] Revers F, García JA . Molecular biology of potyviruses. Adv Virus Res. 2015; 92: 101-99.

[40] Bai S, Cao X, Hu L, et al. Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants. Plant Biotechnol J. 2025; 23: 1153-64.

[41] Sukegawa S, Nureki O, Toki S, et al. Genome editing in rice mediated by miniature size Cas nuclease SpCas12f. Front Genome Ed. 2023; 5: 1-10.

[42] Ishibashi K, Sukegawa S, Endo M, et al. Systemic delivery of engineered compact AsCas12f by a positive-strand RNA virus vector enables highly efficient targeted mutagenesis in plants. Front Plant Sci. 2024; 15: 1-7.

[43] Karmakar S, Panda D, Panda S, et al. A miniature alternative to Cas9 and Cas12: transposon-associated TnpB mediates targeted genome editing in plants. Plant Biotechnol J. 2024; 22: 2950-3.

[44] Bedoya LC, Martínez F, Orzáez D, et al. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiol. 2012; 158: 1130-8.

[45] Thole V, Worland B, Snape JW, et al. The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation. Plant Physiol. 2007; 145: 1211-9.

[46] Pasin F, Bedoya LC, Bernabé-Orts JM, et al. Multiple T-DNA delivery to plants using novel mini binary vectors with compatible replication origins. ACS Synth Biol. 2017; 6: 1962-8.

[47] Bedoya LC, Daròs JA . Stability of tobacco etch virus infectious clones in plasmid vectors. Virus Res. 2010; 149: 234-40.

[48] Fisher DK, Guiltinan MJ . Rapid, efficient production of homozygous transgenic tobacco plants with Agrobacterium tumefaciens: a seed-to-seed protocol. Plant Mol Biol Report. 1995; 13: 278-89.

[49] Ellul P, Garcia-Sogo B, Pineda B, et al. The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculenturn L. Mill.) is genotype and procedure dependent. Theor Appl Genet. 2003; 106: 231-8.