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Massive crossover suppression by CRISPR–Cas-mediated plant chromosome engineering

Nature Plants volume 8, pages 1153–1159 (2022)Cite this article

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Abstract

Recent studies have demonstrated that not only genes but also entire chromosomes can be engineered using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPER-associated protein 9 (Cas9)1,2,3,4,5. A major objective of applying chromosome restructuring in plant breeding is the manipulation of genetic exchange6. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using chromosome restructuring. We were able to induce a heritable inversion of a >17 Mb-long chromosome fragment that contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In single-nucleotide polymorphism marker analysis of the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers within the inverted chromosome region, coupled with a shift of crossovers to the telomeric ends. The few genetic exchanges detected within the inversion all originated from double crossovers. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny.

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Fig. 1: Schematic overview of the CRISPR–Cas9-induced chromosome inversion, WT and inversion junctions, fertility analysis and FISH analysis.
Fig. 2: Fertility and FISH analyses.
Fig. 3: Recombination frequency on chromosome 2 and unambiguously assignable CO events in the inversion line.
Fig. 4: Consequences of the occurrence of subtelomeric, single or double COs in a large chromosome inversion.

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Data availability

The data supporting the findings are available within the article or in Supplementary Information. Source data for Figs. 2b, 3a,b and Extended Data Fig. 3 are provided with this paper.

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Acknowledgements

We thank J. Baumann, N. Schäfter and K. Kumke for their excellent technical assistance and D. Donahey for proofreading the manuscript. This research was funded by the European Research Council (Grant number ERC-2016-AdG_741306 CRISBREED) to H.P.

Author information

Authors and Affiliations

  1. Botanical Institute, Karlsruhe Institute of Technology, Karlsruhe, Germany

    Michelle Rönspies, Carla Schmidt, Patrick Schindele & Holger Puchta

  2. Division of Cancer Research, Department of Thoracic Surgery, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Carla Schmidt

  3. German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Carla Schmidt

  4. Institute of Plant Sciences, Department of Vegetable and Field Crops, Agricultural Research Organization (ARO), Volcani Center, Rishon LeZion, Israel

    Michal Lieberman-Lazarovich

  5. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany

    Andreas Houben

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Contributions

M.R., C.S. and H.P. designed and planned the experiments. M.R., C.S, M.L.-L. and A.H. carried out the experiments. M.R., C.S., P.S, A.H. and H.P. analysed data. M.R. and P.S. created the figures and M.R., P.S., A.H. and H.P. wrote the paper.

Corresponding author

Correspondence to Holger Puchta.

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The authors declare no competing interests.

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Nature Plants thanks Ian Henderson 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 Phenotype analysis of homozygous, hemizygous and WT plants.

Representative pictures were taken from three homozygous, hemizygous and WT plants at 7.5 weeks-old. No phenotypic differences were observed. Scale bars represent 5 cm. Experiments were repeated two times independently with similar results.

Extended Data Fig. 2 Representative siliques from homozygous, hemizygous and WT plants.

We observed empty spaces in place of seeds in several siliques of the hemizygous plants. Pictures were obtained while performing the fertility analysis of homozygous, hemizygous and WT plants with 12 plants per genotype which were randomly selected and 10 randomly selected siliques per plant (Fig. 2b). Scale bars represent 5 mm. Experiments were repeated two times independently with similar results.

Extended Data Fig. 3 Recombination frequency on chromosome 3.

The recombination frequency on chromosome 3 was determined by SNP-based genotyping of 400 plants of the offspring of the Inversion x Ler-1 line (Inv x Ler-1) and the control, Col-0 x Ler-1 for each marker interval (I1-8; Supplementary Table 3). The recombination frequency is presented in cM/Mb. No notable difference in CO distribution between the two crosses on chromosome 3 was detected. Source data are provided as Source Data file.

Source data

Extended Data Fig. 4 Overview of the detected CO events in the Inversion x Ler-1 line on chromosome 2 which cannot be clearly assigned to one of the two chromosomes.

The Col-0 allele is represented by blue bars and the Ler-1 allele by red bars. The detection of both alleles is represented by yellow bars. a, Overview of marker changes which originated from differing single CO on both chromosomes (24). b, Overview of marker changes which originated either from differing single CO on one or both chromosomes or from double CO outside of the inversion or a combination of double CO outside of the inversion with differing single CO (18). c, In 7 samples, marker changes inside the inversion were detected which originated from a combination of a double CO on one chromosome and single CO on one or both chromosomes.

Extended Data Fig. 5 Overview of the detected single CO events in the Col-0 x Ler-1 line on chromosome 2.

The Col-0 allele is represented by blue bars and the Ler-1 allele by red bars. The detection of both alleles is represented by yellow bars. a, Overview of marker changes which originated from single CO on the short arm on one chromosome (29). b, Overview of marker changes which originated from a differing single CO on the short arm on both chromosomes (3; rows 1–3) or two identical single CO on both chromosomes (1; row 4). c, Overview of marker changes which originated from single CO on the long arm on one chromosome (130). d, Overview of marker changes which originated from differing single CO on the long arm of both chromosomes (22). e, Overview of marker changes which originated from differing single CO, one on the long and one of the short arm of either chromosome (19).

Extended Data Fig. 6 Overview of the detected double and/or single CO events in the Col-0 x Ler-1 line on chromosome 2.

The Col-0 allele is represented by blue bars and the Ler-1 allele by red bars. The detection of both alleles is represented by yellow bars. a, Overview of marker changes which originated either from a double CO on one chromosome or from two single CO from two different meioses on one chromosome (24; rows 1–22) and from a combination of either a double CO on one chromosome and a single CO on the other chromosome or two single CO from different meioses on one chromosome and a single CO on the other chromosome (4; rows 23–26). b, Overview of marker changes which originated either from a double CO on one chromosome or from two single CO from two different meioses on one chromosome (60).

Extended Data Fig. 7 Overview of the detected double and/or single CO events in the Col-0 x Ler-1 line on chromosome 2 which could not be unamigously assigned to either of the chromosomes.

The Col-0 allele is represented by blue bars and the Ler-1 allele by red bars. The detection of both alleles is represented by yellow bars. Overview of marker changes which could not be unamigously assigned to either of the chromosomes and originated either from a combination of double CO and single CO on one or both chromosomes or differing single CO from two meioses (39).

Supplementary information

Reporting Summary

Supplementary Tables 1–4

Supplementary Table 1. Mendelian segregation of the inversion line. The progeny of the hemizygous inversion line was genotyped via PCR in the T3 generation by screening for the presence of the inversion-specific and WT junctions. Using the χ² test with the critical value χ² (1; 0.95), it was confirmed that the number of homozygous, heterozygous and WT plants corresponded to Mendel’s laws of segregation. The analysis was repeated two times, with 41 and 37 plants, respectively. Supplementary Table 2. BACs used for FISH analysis. Supplementary Table 3. Overview of the position and sequence of the used SNP markers with corresponding intervals on WT chromosomes 2 (top) and chromosome 3 (bottom). SNPs between Col-0 and Ler-1 are marked in red. The oligonucleotides were modified by adding a fluorophore (HEX/FAM) at the 5’ end and a quencher (BHQ1) at the 3’ end. LNAs integrated into the probe are represented by blue square brackets and a plus sign. Supplementary Table 4. List of oligonucleotides used. Overhangs are represented by lowercase letters.

Source data

Source Data Fig. 2

Raw data for fertility assay.

Source Data Fig. 3

Raw data for calculation of recombination frequency and CO numbers on chromosome 2.

Source Data Extended Data Fig. 3

Raw data for calculation of recombination frequency and CO numbers on chromosome 3.

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Rönspies, M., Schmidt, C., Schindele, P. et al. Massive crossover suppression by CRISPR–Cas-mediated plant chromosome engineering. Nat. Plants 8, 1153–1159 (2022). https://doi.org/10.1038/s41477-022-01238-3

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