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CRISPR–Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis

Nature Plants volume 6, pages 638–645 (2020)Cite this article

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits1,2,3,4. Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus5, we were able to induce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note, translocation frequency was about five times more efficient in the absence of the classical non-homologous end-joining pathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner, fixing or breaking genetic linkages between traits on different chromosomes. Controlled restructuring of plant genomes has the potential to transform plant breeding.

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Fig. 1: Induction of translocations between Chr1 and Chr2 in wild type and the ku70-1 mutant by SaCas9.
Fig. 2: Establishment of lines homozygous for the translocation.
Fig. 3: Analysis of lines carrying the translocation homozygously.
Fig. 4: Induction of heritable translocations between Chr1 and Chr5.

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Data availability

The authors declare that the data supporting the findings are available within the paper and its Supplementary Information, or are available from the corresponding author upon reasonable request.

Code availability

R code for detailed analysis of NGS data is available upon request from the corresponding author.

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Acknowledgements

We thank A. Imhof, K. Siegmund, K. Kumke and C. Gäde for technical assistance with performing experiments, T. Wehrle for assisting with cultivation of the plants in the greenhouse, A. Whitbread for critical reading of the manuscript, T. Zundel for providing an R-based script for bioinformatic analysis of NGS data, A. Neubauer for customizing the script to our needs and M. Lysak for the selection of BACs. This research was funded by the European Research Council (ERC) (Grant number ERC-2016-AdG_741306 CRISBREED) to H.P.

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Authors and Affiliations

  1. Botanical Institute—Molecular Biology and Biochemistry of Plants, Karlsruhe Institute of Technology, Karlsruhe, Germany

    Natalja Beying, Carla Schmidt, Michael Pacher & Holger Puchta

  2. Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, Seeland, Germany

    Andreas Houben

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  1. Natalja Beying
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Contributions

N.B., C.S., M.P. and H.P. designed research; N.B. conducted the research and A.H. performed FISH analysis; N.B., C.S., A.H. and H.P. analysed data; and N.B., A.H. and H.P. wrote the paper.

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Correspondence to Holger Puchta.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Keunsub Lee, Kan Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Detailed analysis of NGS data regarding the repair pattern at the junction sites of TL1-2 in T1 generation.

a, Editing efficiency (percentage of modified reads out of total amount of reads) of SaCas9 at the four different target sites. b, Evaluation of NGS data regarding the deletions at the junction sites. Of this, only the reads carrying a deletion were analysed. The lengths of the deletions were divided into three classes (small: 1-10 bp, middle: 11-50 bp, large: > 50 bp). The deletions at the junction sites in Col-0 were mostly of small or middle size, whereas the ku70 mutant showed bigger amounts of large deletions at both junction sites. c, Quantification of the occurrence of microhomologies (MH, ≥ 2 bp) used for junction formation. We separated the reads into three classes: error-free ligated junctions (green), junctions formed without the use of MHs (orange) or with MHs (yellow). In wild type, most junctions were directly ligated without any mutation induction at the junction sites. Of the reads showing mutations at the junction sites, most junctions were joined without the use of MHs, only a minority of junctions were joined using MHs. In contrast, in the ku70 mutant background nearly no error-free ligation occurred and the prevalent repair pattern at the junctions sites showed the use of MHs for joining. d, e, Detailed representation of the 10 most common reads of both junction sites on sequence level in wild type as well as ku70-1.

Extended Data Fig. 2 Molecular and cytological analysis of two independent plant lines (NBE657#24 and NBE669#30) carrying the TL1-2 homozygously.

a, Schematic overview of PCR amplicons (F1-6) of the translocated chromosome parts. A fragment for amplification of around 2.5 kb every 100 kb was designed for each chromosome arm. b, Cropped gel electrophoresis pictures of PCR amplicons of the translocated chromosome parts. Every 100 kb on the translocated chromosome parts, a band could be detected, indicating no information was lost during translocation formation. As PCR controls, genomic DNA of wild type without translocation formation (positive control) and water (NTC = no template control, negative control) were processed at the same time and loaded on different gels for a better overview. c, By phenotypical comparison of 5 week old plants carrying the TL1-2 to the wild type no differences in growth could be documented. Experiments were repeated two times independently with similar results. d, e, Fertility analysis was conducted by measuring the silique length and counting the number of seeds of five biologically independent samples (n = 5). Barplots show the mean values, error bars as mean ± s.d. For both analysed lines carrying the TL1-2, we could not detect any difference to the wild type.

Source Data

Supplementary information

Supplementary Figure 1

Supplementary Raw Data 1–5 and Tables 1–7.

Reporting Summary

Source data

Source Data Fig. 3

Microscopy pictures TL1-2, unmerged and merged.

Source Data Fig. 4

Microscopy pictures TL1-5, unmerged and merged.

Source Data Extended Data Fig. 2

Unprocessed gel pictures.

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Beying, N., Schmidt, C., Pacher, M. et al. CRISPR–Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis. Nat. Plants 6, 638–645 (2020). https://doi.org/10.1038/s41477-020-0663-x

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