Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9

Abstract

Genome-editing technologies using CRISPR–Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR–Cas reagent delivery methods may be associated with unanticipated genome changes or damage1,2, with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis3. Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR–Cas9 ribonucleoproteins into protoplasts4 or in vitro fertilized zygotes5. However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR–Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency6,7,8,9. Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR–Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR–Cas9 gene editing in plants through virus infection.

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Fig. 1: Engineering SYNV vectors for delivery of CRISPR–Cas9 for N. benthamiana- targeted gene mutagenesis.
Fig. 2: Single and multiplex mutagenesis, and chromosome deletions of endogenous gene loci by SYNV-delivered CRISPR–Cas9.
Fig. 3: Phenotypes and genotypes of M0 plants regenerated from virus-infected leaf tissues.
Fig. 4: Phenotypic segregation patterns of tetra-allelic M0 lines containing non-frameshift PDS homoeoalleles.

Data availability

All data supporting the findings of this study are available in the article or 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 A. O. Jackson and X. Zhou for valuable suggestions and critical reading of the manuscript, and Q.-Y. Shu for assistance in chi-squared test analysis. This work was supported by grants from the National Natural Science Foundation of China (nos. 31870142 and 31671996) and the Natural Science Foundation of Zhejiang Province, China (no. LZ20C140004).

Author information

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Authors

Contributions

Z.L. and X.M. conceived the project and designed the experiments. X.M., X.Z. and H.L. performed the experiments. X.M. and Z.L. analysed the data and interpreted the results. Z.L. and X.M. wrote the manuscript.

Corresponding author

Correspondence to Zhenghe Li.

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Competing interests

Z.L. and X.M. are inventors on a patent application covering the results described in this paper.

Additional information

Peer review information Nature Plants thanks Lanqin Xia, Jian-Kang Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Stability of SYNV vectors after mechanical transmission.

a, GFP fluorescence images of N. benthamiana 16c plants mechanically inoculated with V-Cas9 or V-gGFP1-Cas9. Photographs were taken under long-wavelength UV illumination at 30 days after systemic infection. b, PCR-RE detection of mutation frequencies in plants mechanically infected by V-gGFP1-Cas9. V-Cas9/U and V-Cas9/D denote a V-Cas9-infected plant without or with restriction digestion. M, 1-kb ladder DNA marker. c, RT-PCR detection of gRNA and Cas9 inserts in upper leaves of plants systemically infected by SYNV derivatives. V-WT, wild-type SYNV infection; d, Western blot analysis of the expression of the viral structural proteins, Cas9 and GFP in infected leaf tissues. The Coomassie Brilliant Blue-stained large subunit of RuBisCO (rbcL) severs as protein loading control. Source data

Extended Data Fig. 2 Evaluation of the efficiency and precision of the tRNA-mediated gRNA processing by cRT-PCR.

a, Schematic representation of gRNA circularization and terminal sequence mapping. The divergent arrow pairs denote annealing positions of abutting primers used for cRT-PCR. The hypothesized size of each amplified fragment are indicated. A(n), poly(A) tails with undefined base number. b, cRT-PCR analysis of the gRNA processing efficiency. Arrow denotes DNA fragments corresponding in size to mature gRNA, and asterisk labels the unprocessed 5′ UTR-gRNA-3′ UTR fragment. M, 500-bp ladder marker. c, Sanger sequencing of the gRNA-like cRT-PCR products. The linear sequences are aligned with a canonical gRNA (WT). The sequences of gRNA spacer, scaffold, tRNA 5′ leader, and SYNV UTR are shown in blue, black, purple, and red color letters, respectively. x# shown on the right indicate the number of individual colonies with the identical sequence. Source data

Extended Data Fig. 3 Mutagenesis frequencies induced by tRNA-processed and unprocessed gRNAs.

a, PCR-RE detection of mutagenesis frequency. DNA fragments containing the gGFP2 target site were amplified by PCR from chromosomal DNA extracted from plants infected with SYNV-gRNA-Cas9 or SYNV-tgtRNA-Cas9 and digested by NcoI. Mock/U and Mock/D denote a mock-infected plant without or with restriction digestion. b, Sanger sequencing results of target site mutations induced by SYNV-gRNA-Cas9. Base substitution is italicized, and base deletions are denoted by dashes, with the number of base substitution (s#) and base deletion (d#) indicated on the right of each sequence. Examples of sequencing chromatographs are shown. Source data

Extended Data Fig. 4 Viral symptoms and mutation phenotypes in N. benthamiana.

Plants were agroinoculated with SYNV-tgtRNA-Cas9 vectors targeting different endogenous genomic sites, as indicated on the top of panels. Infected plants were photographs at 20 days after symptom appearance. Mock, mock inoculation; V-WT, infection with wild-type SYNV. Note: despite high mutagenesis frequencies, target gene mutation phenotypes, such as the photobleaching phenotype anticipated from knock-down of PDS, were not observed in the virus-infected plants. This is likely due to the residual activities of the unaltered and altered but non-frameshift PDS alleles, as shown in Fig. 3 in this study. For the N. benthamiana RDR6 and SGS3 gene targets, previous studies have shown that their transgenic RNAi lines exhibit little developmental abnormalities39,40.

Extended Data Fig. 5 Genotyping of M0 plants regenerated from plant tissues infected with SYNV vector targeting the gR6–4 site.

a, Detection of target mutations and viral vector in independent M0 plants by PCR-RE and RT-PCR assays. The size of undigested DNA fragments (indicated by red arrowhead) as well as digestion products are shown along with a 500-bp ladder DNA marker (M). b, Genotyping of M0 plants by Sanger sequencing. M0 plants carrying tetra-allelic mutations are labeled in blue letters. Mutation type: WT, wild-type sequence with no mutation detected; d#, number of bases deleted from target site; i#, number of bases inserted at target site; s#, number of bases substitution at target site. Asterisks denote that both the M0–1 and -9 plants contain a mutated allele with an 8-base deletion that happens to restore the PvuII site (underlined), thus remaining partially susceptible to PvuII digestion as shown in (a). Source data

Extended Data Fig. 6 Genotyping of M0 plants regenerated from plant tissues infected with SYNV vector targeting the gS3–2 site.

a, Detection of target mutations and viral vector in independent M0 plants by PCR-RE and RT-PCR assays. The size of undigested DNA fragments (red arrowhead) and digestion products are labeled, M, 500-bp ladder DNA marker. b, M0 plant genotypes determined by Sanger sequencing. M0 plants with tetra-allelic mutations are labeled in blue letters. Deleted nucleotides are denoted by dash symbols, and inserted nucleotides or substitutions are shown in boldface and italicized letters, respectively. Mutation type: WT, wild-type sequence with no mutation; d#, i#, and s# denote number of bases deleted, inserted, or substituted at target site, respectively. Source data

Extended Data Fig. 7 Segregation of phenotypes and genotypes in M1 and M2 progeny derived from five representative M0 lines bi-allelic for PDS-A and PDS-B.

For each M0 line, the genotypes of one albino and four normal M1 progeny were determined by Sanger sequencing (Supplementary Figure 5). The seeds produced by the normal M1 plants after self-pollination were germinated on MS media plates and the M2 progeny phenotypes counted. P values were calculated by using a chi-squared test of goodness-of-fit to segregation at a 3:1 or 15:1 ratio. 0.1< P <0.5, in good agreement with theoretical segregation ratios; P >0.5, in very good agreement with theoretical segregation ratios. d# and i# denote # of bp deleted and inserted from the target site, respectively, and the in-frame deletion types (d3, d6, and d9) are highlighted in green letters. Ho, homozygote; Bi, bi-allele; n/a, not applicable.

Extended Data Fig. 8 Genotype inheritance of CRISPR–Cas9-mediated PDS gene loci modifications during the M1 to M2 generation.

Five M1 plants homozygous for PDS-A and PDS-B were selected and the genotypes of 10 M2 progeny for each M1 plants were determined by sequencing. d# and i# denote # of bp deleted and inserted from the target site, respectively, and the in-frame deletion types (d3, d6, and d9) are highlighted in green letters. Ho, homozygote. -, virus-free.

Extended Data Fig. 9 Detection of potential off-target effects.

DNA fragments encompassing the gPDS-1 on-target (PDS-A) and 13 potential off-targets (OT1 to OT13; see Supplementary Table 5) were each amplified from the albino M0–11 plant by PCR with specific primers and then subjected to digestion with T7EI and HinfI (if applicable). Lanes 1, 2, and 3 show samples from a mock-infected plant without or with enzyme digestion, and from the M0–11 plant with enzyme digestion, respectively. M, ladder DNA marker. Source data

Extended Data Fig. 10 Absence of SYNV vector in M1 progeny derived from various regenerated M0 lines.

Total RNA extracted from four or five individual M1 plants derived from representative M0 lines were analyzed by RT-PCR with SYNV- and actin-specific primer sets. Inf. indicates an RNA sample extracted from an SYNV-infected plant serving as a positive control. M, 1-kb ladder DNA marker. Source data

Supplementary information

Supplementary Information

Supplementary methods, references, Figs. 1–6 and Tables 1–8.

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Ma, X., Zhang, X., Liu, H. et al. Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9. Nat. Plants (2020). https://doi.org/10.1038/s41477-020-0704-5

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