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One-step genome editing of elite crop germplasm during haploid induction

Nature Biotechnology volume 37, pages 287–292 (2019)Cite this article

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Abstract

Genome editing using CRISPR–Cas9 works efficiently in plant cells1, but delivery of genome-editing machinery into the vast majority of crop varieties is not possible using established methods2. We co-opted the aberrant reproductive process of haploid induction (HI)3,4,5,6 to induce edits in nascent seeds of diverse monocot and dicot species. Our method, named HI-Edit, enables direct genomic modification of commercial crop varieties. HI-Edit was tested in field and sweet corn using a native haploid-inducer line4 and extended to dicots using an engineered CENH3 HI system7. We also recovered edited wheat embryos using Cas9 delivered by maize pollen. Our data indicate that a transient hybrid state precedes uniparental chromosome elimination in maize HI. Edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery. Therefore, edited plants could be used in trait testing and directly integrated into commercial variety development.

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Fig. 1: Elite line editing by maize HI-Edit.
Fig. 2: HI-Edit concept in wide crosses and CENH3-based paternal HI systems.

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Data availability

The authors declare that all data supporting the findings of this study are available in the manuscript and supplementary materials. Sequences (Sanger sequencing) are provided in Supplementary Table 16.

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Acknowledgements

We thank T. Liebler and H.P. Zhou for plant care, C. Fan for Taqman assay design, and K. Cox for sampling. We thank S. Elumalai, S. Nalapalli, H. Zhong, and A. Prairie for transformation and transgenic-plant production. We thank E. Bilyeu, R. Evans, A. Miller, and J. Bower for support with the SNP-based codominant marker analysis. Thanks to D. Bradley and P. Wu for the media preparations, and special thanks to J. Howell, D. Santeramo, and W. Liu for embryo extraction from maize elite inbred haploids, plant care, and data analysis. We thank I. Jepson, E. Dunder and L. Shi for lab space, personnel, and project support. Thanks to C. Leming, M. Dunn, and S. Miles, and M. Edwards for licensing and intellectual property guidance as well as Arabidopsis project support. Thanks to Y. Liu for the images of wheat spikes. Thanks to W. Wang, Y. Jiang, and M. Terry for assistance with plant growth and analysis. Thanks to V. Walbot, F. Altpeter, and K. Wang for reviewing the manuscript.

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Authors and Affiliations

  1. Seeds Research, Syngenta Crop Protection, Research Triangle Park, North Carolina, United States

    Timothy Kelliher, Dakota Starr, Xiujuan Su, Guozhu Tang, Zhongying Chen, Jared Carter, Shujie Dong, Julie Green, Erin Burch, Jamie McCuiston, Weining Gu, Yuejin Sun, Tim Strebe, James Roberts, Nic J. Bate & Qiudeng Que

  2. Department of Plant Biology, University of Georgia, Athens, Georgia, USA

    Dakota Starr

  3. Seeds Research, Syngenta Crop Protection, Enkhuizen, The Netherlands

    Peter E. Wittich

  4. Pairwise Plants, Research Triangle Park, North Carolina, USA

    Nic J. Bate

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  1. Timothy Kelliher
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Contributions

T.K.: experimental design, management, crossing, embryo extraction, analysis and writing. D.S.: wheat and maize crossing, plant sampling, PCR and embryo extraction. X.S.: wheat wide crossing, embryo extraction, sampling and sequencing. G.T.: Arabidopsis experiment management, transformation, crossing, rescue and sequencing. Z.C.: construct design and synthesis. J.C.: event selection and characterization, field-trial management and genotyping. P.E.W.: editing-construct design, event selection and phenotyping VLHP and GW2 mutant lines. S.D.: editing-construct design, maize transformation and plant selection. J.G.: promoter discovery and analysis, and construct design. E.B.: wheat wide crossing, embryo extraction and sampling. J.M.: Taqman trial design and marker analysis for haploid induction and editing. W.G.: Taqman trial design and marker analysis for haploid induction and editing. Y.S.: trial planning and management, plant mutation and sequencing analysis. T.S. and J.R.: plant care, crossing and phenotyping. N.J.B.: experimental design, project leadership, program management and manuscript editing. Q.Q.: idea conception, experimental management and manuscript writing and editing.

Corresponding authors

Correspondence to Timothy Kelliher or Qiudeng Que.

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A patent covering the information in this manuscript was submitted on 4 December 2016.

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Integrated supplementary information

Supplementary Figure 1 The v1 recessive pale leaf phenotype is used to identify haploids.

The temporary virescence occurs in young seedlings, which later recover and develop normally. The maternal parent was v1 / v1 and the paternal haploid inducer parent was V1 / V1. This image is representative hundreds of similar plantlet analyses performed. We used this analysis to identify 95 haploid plants (out of about 3000 progeny total) in the experiment shown in Fig. 1a and Fig. 1b, as well as Supplementary Tables 1, 2, and 3.

Supplementary Figure 2 Flow cytometry results from seedling leaf of control and putative haploids.

a) Control diploid showing the 2N peak at 200, and the 4N peak (endoreduplicated or G2 cells) at 400. b) 125-13 was Cas9+ and appears to be a diploid. c) 125-25 was Cas9+ and appears to be a diploid. d) Control haploid showing the 1N peak at 100, and the 2N (endoreduplicated or G2 cells) at 200. e) 125-15 is a haploid (this plant was not edited). f) 125-18 is an edited haploid. g) 125-33 is an edited haploid. h) 125-29 is an edited haploid. All of the samples (in a-h) were run independently two times with similar results.

Supplementary Figure 3 Sequencing results from five colonies subcloned with the PCR product from the MATL target site in a haploid edited plant.

a, 125-18. b, 125-33. c, 125-29

Supplementary Figure 4 Family tree of the VLHP genes in Chinese spring wheat, barley and maize.

There are two major clades (GT1 clade and VRS1 clade) in this family of genes20. We found putative GT1 orthologues on wheat chromosome 4, which we named TaGT1-2A, -2B, and -2D and a putative barley orthologue that we called HvGT1. There has been a gene duplication of the barley VRS1 orthologue on wheat chromosome 2. We now name these six genes TaVLHP1-2A, -2B, and -2D, and TaVLHP2-2A, -2B, and -2D. In maize, the barley VRS1 gene has also duplicated to become ZmVLHP1 on chromosome 7 and ZmVLHP2 on chromosome 2. The phylogenetic tree was made using an internal version of Greenphyl DB (www.greenphyl.org)38.

Supplementary Figure 5 Ploidy analysis of VLHP2 and GW-2 edited haploids.

a, control diploid. b, JSER44A26 (VLHP2 edited). c, JSER45A63 (VLHP2 edited). d, JSER82A63 (GW2-2 edited).e, JSER82A56 (GW2-2 edited). f, JSER92A27 (GW2-2 edited). All of the samples (in a-f) were run independently two times with similar results.

Supplementary Figure 6 Sequencing of the GW2-2 and VLHP2 target sites in edited haploid mosaics.

GW2-2 (a, b) and VLHP2 (c–e). a, Haploid JSER92A27 target-site PCR gel with two primer sets (GW2-1-F1/ R1 and GW2-1-F1/ R2) confirming this plant as a true mosaic. b, Sequence alignment of the WT GW2-1 sequence with the two sequences recovered from the mosaic plant JSER92A27 – there were two deletions: a 50 bp deletion downstream of the cut site, and a 359 bp deletion upstream. c, Mixed PCR product chromatogram for haploid plant JSER28A12, resolved by ICE as a 19bp deletion (around the cut site) and WT allele. d, Mixed PCR chromatogram for haploid plant JSER41A50, resolved by ICE as a 3bp deletion just after the target site, and a WT allele. e, Mixed PCR chromatogram for haploid plant JSER42A08 (unresolvable by ICE). These PCR reactions, gels and sequences were each run at least two times, with similar results.

Supplementary Figure 7 PCR colony sequencing of GL1 edited haploids.

Each putative edited target site in the Ler edited haploids’ seedling leaf DNA was amplified by PCR and subcloned followed by sequencing of up to 16 colonies to find the target site mutation and to detect any chimerism. a, 12/15 colonies showed an insertion of a T for 1033-A3. b, 16/16 colonies showed an insertion of an A in 1033-C3. C, 9/9 colonies showed a deletion of a G in 1042-G12. d, 9/9 colonies showed a deletion of a GA at the target site in plant 1042-G10. e, 9/9 colonies showed a deletion of a TG at the target site in plant 1045-E3.

Supplementary Figure 8 Ploidy analysis histograms of Arabidopsis haploid inducer parents and edited haploid progeny.

a, Haploid inducer parent USR01424135. b, Haploid inducer parent USR01424136. c, Haploid inducer parent USR01431609. d, Edited haploid 1033-C3. e, Edited haploid 1033-A3. f, Edited haploid 1041-H12. The 1N and 2N peaks are marked in (a) and (d). These samples shown here (a-f) were run two times, with similar results.

Supplementary Figure 9 Ploidy diagrams of diploid wheat and JSWER30A22 wheat edited haploid with confirmed 97-bp deletion in TaGT1-4B.

a, Diploid wheat. b, JSWER30A22 wheat. The 2N peak (AABBDD) is located at 400. The 1N (ABD) haploid peak is located at 200. Both samples were run twice with similar results.

Supplementary Figure 10 Expression of Cas9 under different promoters.

Cas9 pollen expression was ~100x higher the three pollen-specific promoters (PRF3, EXPB1, and EXPB2) compared to the MATL promoter, which is about the same level as the sugar cane ubiquitin promoter expressed in seedling leaves. Each sample type represents four, (prSoUbi4), five (prMATL) or seven (all others) biological replicates; their average is represented by the center value, and the standard deviation is represented by the error bars.

Supplementary Figure 11 Negative PCR assays for putatively edited wheat haploids.

Successful amplification of unedited genes (Table S11) combined with multiple negative results for edited gene PCRs suggests that large deletions occurred. Each PCR reaction and gel was run twice with similar results. a, Negative PCR assay for the putative edited haploid 447-G8 compared to unedited control. This assay queried 384 nt upstream of the PCR binding site. In the Taqman qPCR assay, the VLHP2-2A well had strong signal for the control ADH1 assay (Ct = 24.5) and weak amplification of VLHP2-2A (Table S11); the score of 0.71 may be due to non-specific amplification of the VLHP2-2D allele, which has some overlap due to shared primers (see Table S13). A gel-based PCR for VLHP-2B and VLHP-2D produced bands, and direct sequencing showed that there was a WT sequence, but for the putatively edited -2A allele, there was no band (assay 2A-F with 2A-R, see Table S12). That first PCR assay queried -178 bp to +227 bp from the cut site. Additional PCR assays were designed. Primer 2A-F2 (+6 bp) and 2A-R (+227 bp) gave a band with WT sequence 3’ of the cut site. A PCR assay using primer 2A-F1 (-384 bp) through +50 bp (2A-R2) did not amplify in 447-G8 but did amplify WT in a positive control. This is shown here in Fig. S11a. This suggests a large deletion occurred 5’ of the cut site, leaving 3’ sequence intact. Ten forward primers (2A-F3 through 2A-F12) were designed upstream, spaced every few hundred bp, all the way until -3.9 kb upstream of the cut site. These were paired with the working 2A-R primer at +227 bp. No PCR products were obtained from any of these new pairs. Taken together, this suggests that a 5’ deletion has occurred, removing a large amount of DNA starting at the target site and proceeding upstream – leading to a deletion of at least several kb. b, Negative PCR assays for four haploids edited in VLHP2-2B, spanning the target site by ~1400 bp (383bp upstream and 1032bp downstream), compared to unedited control. In the Taqman qPCR assay, we saw positive (WT) amplification of VLHP-2A and VLHP-2D, as well as strong amplification for the in-well ADH1 control assays (Ct = 22-24, Table S11). The Gel-based PCR for VLHP2-2B and VLHP2-2D produced PCR bands and direct sequencing showed that there was a WT sequence, but VLHP2-2A did not produce a band. That assay queried -177 bp to +253 bp from the cut site (primers 2B-F and 2B-R). Two additional primer pairs were then designed to query either side of the editing site, to determine which of the primer binding sites were deleted. No bands were found in the gel for 440-D3, 440-A5, and 456-G9. A weak band was produced for the reaction amplifying 3’ of the cut site 440-B3. However, sequencing showed a 100% match to the D genome, indicating non-specific amplification. We then tried to amplify across what we presumed to be a large deletion, designing additional primer pairs up to 1415 bp flanking the target site. We did not recover any PCR product from these reactions, even though the assays successfully amplified the control (unedited) sample, shown below. We conclude that all four plants (440-B3, 440-D3, 440-A5, and 456-G9) have large deletions at the 2B target site.

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Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1–15

Reporting Summary

Supplementary Table 16

Sanger sequences

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Kelliher, T., Starr, D., Su, X. et al. One-step genome editing of elite crop germplasm during haploid induction. Nat Biotechnol 37, 287–292 (2019). https://doi.org/10.1038/s41587-019-0038-x

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