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Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat

Nature Plants volume 11, pages 1252–1259 (2025)Cite this article

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Abstract

CRISPR–Cas genome editing technology is a cutting-edge strategy for crop breeding. However, the delivery of genome-editing reagents remains to be a technological bottleneck in monocot plants1. Here we engineered barley yellow striate mosaic virus (BYSMV) into a negative-strand RNA virus-based vector system2 for delivery of both Cas9 and single guide RNA to achieve heritable gene editing in different wheat cultivars. We found that fusion of a mobile transfer RNA sequence3 to the Cas9 messenger RNA and single guide RNAs could deliver them into the growth points of axillary meristems to achieve gene editing before tiller generation. The resulting nascent tillers contained simultaneous mutations in the three homoeoalleles. Moreover, the progeny seedlings are virus-free and harbour bi-allelic or homozygous mutations. Given BYSMV infects 26 monocot species4, the BYSMV delivery system could have wide applicability for achieving highly efficient, non-transgenic and less genotype-dependent heritable genome editing, thereby facilitating genomic studies and crops breeding.

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Fig. 1: Induction of mutant tillers of hexaploid wheat infected with BY–Cas9T–sgRNATaPDS-T.
Fig. 2: Heritable gene editing from edited tillers of wheat plants infected with BY–Cas9T–sgRNATaPDS-T.
Fig. 3: Heritable gene editing in functional genes without visible phenotype using the BY–Cas9T–sgRNAT delivery system.

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Data availability

All the amplicon sequencing data are available in the National Center for Biotechnology Information Sequence Read Archive (PRJNA1260673). All data supporting the findings of this study are available within the Article and its Supplementary Information or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank S. P. Dinesh-Kumar (University of California, Davis) and S.-W. Ding (University of California, Riverside) for help with editing the manuscript. We are grateful for helpful discussion from our colleagues J. Yu, Y. Zhang and J. Lai. We thank D. Wang (Henan Agricultural University) for providing seeds of different wheat varieties and Q. Chen (China Agricultural University) for providing the pBUE411 plasmid. Research in Xian-Bing Wang’s laboratory is supported by National Natural Science Foundation of China (32425046 to X.-B.W.), National Key Research and Development Program of China (2023YFD1400300 to X.-B.W.) and Pinduoduo-China Agricultural University Research Fund (PC2023B01008 to X.-B.W.).

Author information

Author notes

  1. These authors contributed equally: Ji-Hui Qiao, Ying Zang.

Authors and Affiliations

  1. State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing, China

    Ji-Hui Qiao, Ying Zang, Qiang Gao, Xin-Wen Zhang, Weiyao Hu, Dawei Li & Xian-Bing Wang

  2. College of Plant Protection, China Agricultural University, Beijing, China

    Qiang Gao, Ying Wang & Chenggui Han

  3. State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, China Agricultural University, Beijing, China

    Shengnan Liu

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  1. Ji-Hui Qiao
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Contributions

J.-H.Q., Y.Z., Q.G. and X.-W.Z. performed the experiments. J.-H.Q., S.L. and W.H. analysed the data. J.-H.Q. and X.-B.W. wrote the manuscript, assisted by D.L., C.H. and Y.W. All authors discussed the results and contributed to data interpretation.

Corresponding author

Correspondence to Xian-Bing Wang.

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Competing interests

X.-B.W., J.-H.Q., Y.Z., Q.G., Y.W., C.H. and D.L. (all from China Agricultural University) are co-inventors of Chinese patent application number 202411608574.7 based on the results described in this study. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Somatic gene editing induced by the BY-Cas9-sgRNATaPDS recombinant virus in wheat.

a, Schematic diagrams of the pBY-RFP, pBY-Cas9-sgRNATaPDS, pVSRs, and pNPL plasmids. The Cas9 gene and gRNA flanked with N/P and P6/M junction regions were inserted into the gene junctions of N/P and P6/M genes of BYSMV, respectively. The viral suppressors of RNA silencing (VSRs) include tomato bushy stunt virus p19, tobacco etch virus HC-Pro, and barley stripe mosaic virus γb. The sgRNATaPDS targets wheat phytoene desaturase (TaPDS). b, Illustration of the procedure for rescuing recombinant BYSMV viruses in N. benthamiana leaves, small brown planthoppers (SBPHs), and wheat plants, sequentially. Rice seedlings are not host plants of BYSMV, thus serving as feeding plants of SBPHs for virus incubation. c, Symptoms and RFP fluorescence of wheat plants infected with BY-RFP or BY-Cas9-sgRNATaPDS. Representative infected plants were photographed under a LUYOR-3415RG handheld lamp at 15 days post-infection (d.p.i.). Scale bars, 2 cm. d, Immunoblotting analyses of the Cas9 and viral matrix (M) proteins in systemically infected leaves of panel c. GAPC2 served as a loading control. The Cas9-Flag, M, and GAPC2 proteins were detected with anti-Flag, anti-M, and anti-GAPC2 antibodies, respectively.The experiment was repeated three times. e, Mutation frequencies in three homeoalleles of TaPDS in systemically wheat leaves infected with BY-Cas9-sgRNATaPDS. The editing efficiencies were examined by high-throughput sequencing (HTS) of PCR amplicons spanning the TaPDS target site. The experiments were repeated 3 times (n = 3). Data represents individual data points and mean ± SD. P-values were derived from a two-tailed Student’s t-test. f, Mutation patterns and frequencies at the TaPDS target sites from panel e. The protospacer adjacent motif (PAM) sequence and targeted sequence were highlighted by the red and blue lines, respectively. Indels were indicated as red dashes or letters. WT indicates the wild-type sequence.

Source data

Extended Data Fig. 2 Fusion of the tRNAMet-like sequence to the Cas9 and sgRNA increases virus infection and gene editing in somatic cells.

a, RFP fluorescence of N. benthamiana leaves infected with BY-RFP, BY-Cas9-sgRNATaPDS, or BY-Cas9T-sgRNATaPDS-T at 14 d.p.i. Both the Cas9 and sgRNATaPDS were fused with the tRNAMet sequence as shown in Fig. 1a. Scale bars, 500 μm. The experiment was repeated three times. b, Disease symptoms and RFP fluorescence of wheat plants infected with BY-RFP, BY-Cas9-sgRNATaPDS, or BY-Cas9T-sgRNATaPDS-T. Plants were photographed at 15 d.p.i. under visible light and a LUYOR-3415RG handheld lamp. Scale bars, 5 cm. c, Immunoblotting analyses of Cas9 and viral M protein in systemically infected leaves of plants shown in c. GAPC2 served as a loading control. The Cas9-Flag, M, and GAPC2 proteins were detected with anti-Flag, anti-M, and anti-AtGAPC2 antibodies, respectively. The relative accumulation of Cas9 and M proteins were calculated from band densities using the software ImageJ. The relative values represent means ± SD (n = 3). d, Mutation patterns and frequencies at the PDS target sites. The PAM sequence and targeted sequence were highlighted by the red and blue lines, respectively. Indels were indicated as red dashes or letters. WT indicates wild-type sequences. e, Total editing efficiencies in somatic cells of wheat plants infected with BY-Cas9-sgRNATaPDS or BY-Cas9T-sgRNATaPDS-T. Data represents individual data points and mean ± SD of six independent experiments (n = 6). P-values were derived from a two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 Disease symptom and RFP fluorescence of wheat plants infected with BY-RFP or BY-Cas9T-sgRNATaPDS-T at 150 d.p.i. (a) and 30 d.p.i. (b), respectively.

Representative plants were photographed under visible light and a LUYOR-3415RG handheld lamp. Scale bars, 2 cm.

Extended Data Fig. 4 Detection of virus and Cas9/sgRNA in M1 progeny seedlings through RT-PCR and immunoblotting.

a, Detection of Cas9-T mRNA, sgRNATaPDS-T and BYSMV genomic RNA in M1 progeny seedlings by RT-PCR. The M0 infected and mock wheat leaves served as positive and negative controls, respectively. TaActin served as an endogenous control. The experiment was repeated in M1 seedlings three times. b, Detection of the Cas9 and BYSMV M proteins in M0 infected wheat plants and M1 progeny seedlings by immunoblotting. GAPC2 served as protein loading control. The experiment was repeated in M1 seedlings three times.

Source data

Extended Data Fig. 5 Somatic gene editing in different wheat cultivars infected with BY-Cas9T-sgRNATaPDS-T.

a, Disease symptoms and RFP fluorescence of different wheat cultivars infected with BY-Cas9T-sgRNATaPDS-T. Scale bars, 2 cm. b, Bleached symptoms and RFP fluorescence of BY-Cas9T-sgRNATaPDS-T infected leaves from different wheat cultivars. Scale bars, 1 cm. c, Total editing efficiencies in somatic cells of different wheat cultivars infected with BY-Cas9T-sgRNATaPDS-T. Data represents individual data points and mean ± SD of three independent experiments (n = 3).

Source data

Extended Data Fig. 6 Induction of gene editing tillers in different wheat cultivars infected with BY-Cas9T-sgRNATaPDS-T.

a, Phenotypes of newly emerged tillers from different wheat cultivars infected with BY-Cas9T-sgRNATaPDS-T. Representative plants with albino tillers were photographed at 70 d.p.i. Scale bars, 2 cm. b, Mutation patterns of the TaPDS target in albino tillers from different wheat cultivars. Gene editing patterns were analyzed in three homeoalleles of TaPDS. WT indicates the wild-type sequence. c, Genotyping of the target TaPDS site in the M1 progeny seedlings from different wheat cultivars. Arrow heads indicate the detected seedlings. Scale bars, 2 cm.

Source data

Extended Data Fig. 7 Induction of somatic gene editing in different target genes using BYSMV editing systems.

a, Schematic diagram of the recombinant BY-Cas9T-sgRNATaeIF4E-T genome structures. The sgRNA targets the genes encoding wheat Eukaryotic translation initiation factor 4E (TaeIF4E). The tRNAMet-like structures (TLS) sequence acting as a mobile element was fused to the C termini of the Cas9 and sgRNA genes. b, Phenotype and red fluorescence of wheat plants infected with BY-Cas9T-sgRNATaSDN1-T or BY-Cas9T-sgRNATaeIF4E-T. Representative plants infected with viral editing vectors targeting TaSDN1 gene and TaeIF4E gene were photographed at 15 d.p.i. under visible light and a LUYOR-3415RG handheld lamp. Scale bars, 2 cm. c, Editing efficiencies of the different targets in systemically infected leaves of wheat plants from panel a. Editing efficiencies were determined by high throughput sequencing (HTS) of PCR amplicons spanning the target sites. The experiments were repeated 3 times (n = 3). Data represents individual data points and mean ± SD. P-values were derived from a two-tailed Student’s t-test. d-e, Different somatic mutation types at TaSDN1 and TaeIF4E target sites induced by infection of BY-Cas9T-sgRNATaSDN1-T and BY-Cas9T-sgRNATaeIF4E-T, respectively. The PAM sequence target sequences are indicated by the red line and the blue line, respectively. Mutations are indicated as red dashes or letters. WT indicates the wild-type sequence.

Source data

Extended Data Fig. 8 Induction of gene editing wheat tillers in different target genes using BYSMV editing systems.

a, Numbers of infected wheats and the frequencies of mutant plants. At 60 d.p.i., all the virus-free tillers from one infected wheat plant were mixed and subjected to mutation detection independently for calculating the frequencies of infected plants with mutated tillers. b, Mutation types at TaSDN1 and TaeIF4E target sites in chimeric or heterozygous tillers induced by infection of BY-Cas9T-sgRNATaSDN1-T and BY-Cas9T-sgRNATaeIF4E-T, respectively. c, Representative phenotype and mutations of BY-Cas9T-sgRNATaeIF4E-T-infected wheat plants at approximately 210 d.p.i. Mutation patterns of 9 mutated tillers with bi-allelic or homozygous mutations were shown on the right panels. Scale bar, 5 cm.

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Qiao, JH., Zang, Y., Gao, Q. et al. Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat. Nat. Plants 11, 1252–1259 (2025). https://doi.org/10.1038/s41477-025-02023-8

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