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CRISPR–Cas-mediated chromosome engineering for crop improvement and synthetic biology

Abstract

Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR–Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR–Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant chromosome, but also of novel plant species.

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Fig. 1: Enhancement of meiotic crossovers by modulation of crossover control factors.
Fig. 2: Control of genetic exchange by targeted induction of crossovers and inversions.
Fig. 3: Control of genetic linkages by targeted induction of reciprocal translocations.
Fig. 4: Future perspective on chromosome engineering in plants.

References

  1. Bailey-Serres, J., Parker, J. E., Ainsworth, E. A., Oldroyd, G. E. D. & Schroeder, J. I. Genetic strategies for improving crop yields. Nature 575, 109–118 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Wolter, F., Schindele, P. & Puchta, H. Plant breeding at the speed of light: the power of CRISPR/Cas to generate directed genetic diversity at multiple sites. BMC Plant Biol. 19, 176 (2019).

    PubMed  PubMed Central  Google Scholar 

  3. Lewis, R. S. & Rose, C. Agronomic performance of tobacco mosaic virus-resistant tobacco lines and hybrids possessing the resistance gene N introgressed on different chromosomes. Crop Sci. 50, 1339–1347 (2010).

    CAS  Google Scholar 

  4. Li, J., Chitwood, J., Menda, N., Mueller, L. & Hutton, S. F. Linkage between the I-3 gene for resistance to Fusarium wilt race 3 and increased sensitivity to bacterial spot in tomato. Theor. Appl. Genet. 131, 145–155 (2018).

    CAS  PubMed  Google Scholar 

  5. Zhang, Y., Pribil, M., Palmgren, M. & Gao, C. A CRISPR way for accelerating improvement of food crops. Nat. Food 1, 200–205 (2020).

    Google Scholar 

  6. Zaidi, S. S.-E.-A., Mahas, A., Vanderschuren, H. & Mahfouz, M. M. Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants. Genome Biol. 21, 289 (2020).

    PubMed  PubMed Central  Google Scholar 

  7. Schindele, A., Dorn, A. & Puchta, H. CRISPR/Cas brings plant biology and breeding into the fast lane. Curr. Opin. Biotechnol. 61, 7–14 (2020).

    CAS  PubMed  Google Scholar 

  8. Atkins, P. A. & Voytas, D. F. Overcoming bottlenecks in plant gene editing. Curr. Opin. Plant Biol. 54, 79–84 (2020).

    CAS  PubMed  Google Scholar 

  9. Zhang, Y., Malzahn, A. A., Sretenovic, S. & Qi, Y. The emerging and uncultivated potential of CRISPR technology in plant science. Nat. Plants 5, 778–794 (2019).

    PubMed  Google Scholar 

  10. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Puchta, H. The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J. Exp. Bot. 56, 1–14 (2004).

    PubMed  Google Scholar 

  12. Schmidt, C., Schindele, P. & Puchta, H. From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas. aBIOTECH 1, 21–31 (2020).

    Google Scholar 

  13. Rönspies, M., Schindele, P. & Puchta, H. CRISPR/Cas-mediated chromosome engineering: opening up a new avenue for plant breeding. J. Exp. Bot. 72, 177–183 (2021).

    PubMed  Google Scholar 

  14. Thompson, S. L. & Compton, D. A. Chromosomes and cancer cells. Chromosome Res. 19, 433–444 (2011).

    PubMed  PubMed Central  Google Scholar 

  15. Park, C.-Y., Sung, J. J. & Kim, D.-W. Genome editing of structural variations: modeling and gene correction. Trends Biotechnol. 34, 548–561 (2016).

    CAS  PubMed  Google Scholar 

  16. Brunet, E. & Jasin, M. Induction of chromosomal translocations with CRISPR-Cas9 and other nucleases: understanding the repair mechanisms that give rise to translocations. Adv. Exp. Med. Biol. 1044, 15–25 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Hasty, P. & Montagna, C. Chromosomal rearrangements in cancer: detection and potential causal mechanisms. Mol. Cell. Oncol. 1, e29904 (2014).

    PubMed  PubMed Central  Google Scholar 

  18. Lakich, D., Kazazian, H. H. Jr, Antonarakis, S. E. & Gitschier, J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat. Genet. 5, 236–241 (1993).

    CAS  PubMed  Google Scholar 

  19. Bondeson, M. L. et al. Inversion of the IDS gene resulting from recombination with IDS-related sequences is a common cause of the Hunter syndrome. Hum. Mol. Genet. 4, 615–621 (1995).

    CAS  PubMed  Google Scholar 

  20. Small, K., Iber, J. & Warren, S. T. Emerin deletion reveals a common X-chromosome inversion mediated by inverted repeats. Nat. Genet. 16, 96–99 (1997).

    CAS  PubMed  Google Scholar 

  21. Park, C.-Y., Lee, D. R., Sung, J. J. & Kim, D.-W. Genome-editing technologies for gene correction of hemophilia. Hum. Genet. 135, 977–981 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kirkpatrick, M. & Barton, N. Chromosome inversions, local adaptation and speciation. Genetics 173, 419–434 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Fransz, P. et al. Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana. Plant J. 88, 159–178 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Harewood, L. & Fraser, P. The impact of chromosomal rearrangements on regulation of gene expression. Hum. Mol. Genet. 23, R76–R82 (2014).

    CAS  PubMed  Google Scholar 

  25. Martin, G. et al. Chromosome reciprocal translocations have accompanied subspecies evolution in bananas. Plant J. 104, 1698–1711 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wellenreuther, M. & Bernatchez, L. Eco-evolutionary genomics of chromosomal inversions. Trends Ecol. Evol. 33, 427–440 (2018).

    PubMed  Google Scholar 

  27. Beying, N., Schmidt, C., Pacher, M., Houben, A. & Puchta, H. CRISPR–Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis. Nat. Plants 6, 638–645 (2020).

    CAS  PubMed  Google Scholar 

  28. Schmidt, C. et al. Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering. Nat. Commun. 11, 4418 (2020).

    PubMed  PubMed Central  Google Scholar 

  29. Schwartz, C. et al. CRISPR–Cas9-mediated 75.5-Mb inversion in maize. Nat. Plants 6, 1427–1431 (2020).

    CAS  PubMed  Google Scholar 

  30. Taagen, E., Bogdanove, A. J. & Sorrells, M. E. Counting on crossovers: controlled recombination for plant breeding. Trends Plant Sci. 25, 455–465 (2020).

    CAS  PubMed  Google Scholar 

  31. Wang, Y. & Copenhaver, G. P. Meiotic recombination: mixing it up in plants. Annu. Rev. Plant Biol. 69, 577–609 (2018).

    CAS  PubMed  Google Scholar 

  32. Lambing, C., Franklin, F. C. H. & Wang, C.-J. R. Understanding and manipulating meiotic recombination in plants. Plant Physiol. 173, 1530–1542 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Bergerat, A. et al. An atypical topoisomerase II from archaea with implications for meiotic recombination. Nature 386, 414–417 (1997).

    CAS  PubMed  Google Scholar 

  34. Crismani, W. et al. FANCM limits meiotic crossovers. Science 336, 1588–1590 (2012).

    CAS  PubMed  Google Scholar 

  35. Hartung, F., Suer, S. & Puchta, H. Two closely related RecQ helicases have antagonistic roles in homologous recombination and DNA repair in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 104, 18836–18841 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Séguéla-Arnaud, M. et al. Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM. Proc. Natl Acad. Sci. USA 112, 4713–4718 (2015).

    PubMed  PubMed Central  Google Scholar 

  37. Dorn, A. et al. The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates. PLoS Genet. 14, e1007674 (2018).

    PubMed  PubMed Central  Google Scholar 

  38. Séguéla-Arnaud, M. et al. RMI1 and TOP3α limit meiotic CO formation through their C-terminal domains. Nucleic Acids Res. 45, 1860–1871 (2017).

    PubMed  Google Scholar 

  39. de Maagd, R. A. CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid. Plant Biotechnol. J. 18, 805–813 (2020).

    CAS  PubMed  Google Scholar 

  40. Girard, C. et al. AAA-ATPase FIDGETIN-LIKE 1 and helicase FANCM antagonize meiotic crossovers by distinct mechanisms. PLoS Genet. 11, e1005369 (2015).

    PubMed  PubMed Central  Google Scholar 

  41. Fernandes, J. B. et al. FIGL1 and its novel partner FLIP form a conserved complex that regulates homologous recombination. PLoS Genet. 14, e1007317 (2018).

    PubMed  PubMed Central  Google Scholar 

  42. Zhang, P. et al. The rice AAA-ATPase OsFIGNL1 is essential for male meiosis. Front. Plant Sci. 8, 1639 (2017).

    PubMed  PubMed Central  Google Scholar 

  43. Mieulet, D. et al. Unleashing meiotic crossovers in crops. Nat. Plants 4, 1010–1016 (2018).

    CAS  PubMed  Google Scholar 

  44. Pyatnitskaya, A., Borde, V. & de Muyt, A. Crossing and zipping: molecular duties of the ZMM proteins in meiosis. Chromosoma 128, 181–198 (2019).

    CAS  PubMed  Google Scholar 

  45. Ziolkowski, P. A. et al. Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination. Genes Dev. 31, 306–317 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Serra, H. et al. Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis. Proc. Natl Acad. Sci. USA 115, 2437–2442 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Wang, K., Wang, C., Liu, Q., Liu, W. & Fu, Y. Increasing the genetic recombination frequency by partial loss of function of the synaptonemal complex in rice. Mol. Plant 8, 1295–1298 (2015).

    CAS  PubMed  Google Scholar 

  48. Fernandes, J. B., Séguéla-Arnaud, M., Larchevêque, C., Lloyd, A. H. & Mercier, R. Unleashing meiotic crossovers in hybrid plants. Proc. Natl Acad. Ssci. USA 115, 2431–2436 (2018).

    CAS  Google Scholar 

  49. Hartung, F., Suer, S., Knoll, A., Wurz-Wildersinn, R. & Puchta, H. Topoisomerase 3α and RMI1 suppress somatic crossovers and are essential for resolution of meiotic recombination intermediates in Arabidopsis thaliana. PLoS Genet. 4, e1000285 (2008).

    PubMed  PubMed Central  Google Scholar 

  50. Mannuss, A. et al. RAD5A, RECQ4A, and MUS81 have specific functions in homologous recombination and define different pathways of DNA repair in Arabidopsis thaliana. Plant Cell 22, 3318–3330 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Schröpfer, S., Kobbe, D., Hartung, F., Knoll, A. & Puchta, H. Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis. Nucleic Acids Res. 42, 1684–1697 (2014).

    PubMed  Google Scholar 

  52. Knoll, A. et al. The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis. Plant Cell 24, 1448–1464 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Peciña, A. et al. Targeted stimulation of meiotic recombination. Cell 111, 173–184 (2002).

    PubMed  Google Scholar 

  54. Sarno, R. et al. Programming sites of meiotic crossovers using Spo11 fusion proteins. Nucleic Acids Res. 45, e164 (2017).

    PubMed  PubMed Central  Google Scholar 

  55. Yelina, N. E., Gonzalez-Jorge, S., Hirsz, D., Yang, Z. & Henderson, I. R. CRISPR targeting of MEIOTIC-TOPOISOMERASE VIB-dCas9 to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis. Preprint at bioRxiv https://doi.org/10.1101/2021.02.01.429210 (2021).

  56. Filler Hayut, S., Melamed Bessudo, C. & Levy, A. A. Targeted recombination between homologous chromosomes for precise breeding in tomato. Nat. Commun. 8, 15605 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Ben Shlush, I. et al. CRISPR/Cas9 induced somatic recombination at the CRTISO locus in tomato. Genes 12, 59 (2020).

    PubMed  PubMed Central  Google Scholar 

  58. Davis, L., Khoo, K. J., Zhang, Y. & Maizels, N. POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks. Proc. Natl Acad. Sci. USA 117, 22900–22909 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Cheong, T.-C., Blasco, R. B. & Chiarle, R. The CRISPR/Cas9 System as a tool to engineer chromosomal translocation in vivo. Adv. Exp. Med. Biol. 1044, 39–48 (2018).

    CAS  PubMed  Google Scholar 

  60. Torres-Ruiz, R. et al. Efficient recreation of t(11;22) EWSR1-FLI1+ in human stem cells Using CRISPR/Cas9. Stem Cell Rep. 8, 1408–1420 (2017).

    CAS  Google Scholar 

  61. Torres, R. et al. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system. Nat. Commun. 5, 3964 (2014).

    CAS  PubMed  Google Scholar 

  62. Lagutina, I. V. et al. Modeling of the human alveolar rhabdomyosarcoma Pax3–Foxo1 chromosome translocation in mouse myoblasts using CRISPR–Cas9 nuclease. PLoS Genet. 11, e1004951 (2015).

    PubMed  PubMed Central  Google Scholar 

  63. Choi, P. S. & Meyerson, M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5, 3728 (2014).

    CAS  PubMed  Google Scholar 

  64. Luo, J., Sun, X., Cormack, B. P. & Boeke, J. D. Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast. Nature 560, 392–396 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Shao, Y. et al. Creating a functional single-chromosome yeast. Nature 560, 331–335 (2018).

    CAS  PubMed  Google Scholar 

  66. Fleiss, A. et al. Reshuffling yeast chromosomes with CRISPR/Cas9. PLoS Genet. 15, e1008332–e1008332 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Yadav, V., Sun, S., Coelho, M. A. & Heitman, J. Centromere scission drives chromosome shuffling and reproductive isolation. Proc. Natl Acad. Sci. USA 117, 7917–7928 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Jayakodi, M. et al. The barley pan-genome reveals the hidden legacy of mutation breeding. Nature 588, 284–289 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Walkowiak, S. et al. Multiple wheat genomes reveal global variation in modern breeding. Nature 588, 277–283 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Crow, T. et al. Gene regulatory effects of a large chromosomal inversion in highland maize. PLoS Genet. 16, e1009213 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Schmidt, C., Pacher, M. & Puchta, H. Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system. Plant J. 98, 577–589 (2019).

    CAS  PubMed  Google Scholar 

  72. Fransz, P. F. et al. Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region. Cell 100, 367–376 (2000).

    CAS  PubMed  Google Scholar 

  73. Drouaud, J. et al. Variation in crossing-over rates across chromosome 4 of Arabidopsis thaliana reveals the presence of meiotic recombination “hot spots”. Genome Res. 16, 106–114 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Zhang, Y., Cheng, Z., Ma, J., Xian, F. & Zhang, X. Characteristics of a novel male–female sterile watermelon (Citrullus lanatus) mutant. Sci. Horticulturae 140, 107–114 (2012).

    Google Scholar 

  75. Steinert, J., Schiml, S., Fauser, F. & Puchta, H. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J. 84, 1295–1305 (2015).

    CAS  PubMed  Google Scholar 

  76. Schindele, P. & Puchta, H. Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing. Plant Biotechnol. J. 18, 1118–1120, https://doi.org/10.1111/pbi.13275 (2020).

    Article  PubMed  Google Scholar 

  77. Wolter, F., Klemm, J. & Puchta, H. Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staphylococcus aureus. Plant J. 94, 735–746 (2018).

    CAS  PubMed  Google Scholar 

  78. Mandáková, T. & Lysak, M. A. Post-polyploid diploidization and diversification through dysploid changes. Curr. Opin. Plant Biol. 42, 55–65 (2018).

    PubMed  Google Scholar 

  79. Liu, Y. et al. Rapid birth or death of centromeres on fragmented chromosomes in maize. Plant Cell 32, 3113–3123 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Wimmer, E., Mueller, S., Tumpey, T. M. & Taubenberger, J. K. Synthetic viruses: a new opportunity to understand and prevent viral disease. Nat. Biotechnol. 27, 1163–1172 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Coradini, A. L. V., Hull, C. B. & Ehrenreich, I. M. Building genomes to understand biology. Nat. Commun. 11, 6177 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Dawe, R. K. Charting the path to fully synthetic plant chromosomes. Exp. Cell. Res. 390, 111951 (2020).

    CAS  PubMed  Google Scholar 

  83. Liu, J. et al. Genome-scale sequence disruption following biolistic transformation in rice and maize. Plant Cell 31, 368–383 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Zhang, H. et al. Stable integration of an engineered megabase repeat array into the maize genome. Plant J. 70, 357–365 (2012).

    PubMed  Google Scholar 

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Acknowledgements

We thank D. Donahey for proofreading the manuscript. This work was supported by the European Research Council (Advanced grant: ERC-2016-AdG_741306 CRISBREED).

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All authors wrote the manuscript. A.D. and P.S. designed the figures. P.S. created the figures.

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Correspondence to Holger Puchta.

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Rönspies, M., Dorn, A., Schindele, P. et al. CRISPR–Cas-mediated chromosome engineering for crop improvement and synthetic biology. Nat. Plants 7, 566–573 (2021). https://doi.org/10.1038/s41477-021-00910-4

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