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CRISPR–Cas9-mediated chromosome engineering in Arabidopsis thaliana

Nature Protocols volume 17, pages 1332–1358 (2022)Cite this article

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Abstract

The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.

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Fig. 1: Examples of chromosomal rearrangements generated by chromosome engineering.
Fig. 2: Screening strategy for the identification of chromosomal rearrangements and establishment of homozygous lines.
Fig. 3: Principle of a probe-based duplex assay to measure translocation efficiency.
Fig. 4: Analysis of a simplex thermal gradient PCR using the QuantaSoft Analysis Pro Software.
Fig. 5: Primer combinations for PCR screenings, exemplary shown for an inversion event.

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Data availability

The data shown in Tables 3 and 4 are available in the supporting primary research papers (refs. 5,6, respectively). Figure 4 represents data that have not been previously published and are provided as Supplementary Data 1.

References

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

    Article  CAS  Google Scholar 

  2. Rönspies, M., Dorn, A., Schindele, P. & Puchta, H. CRISPR–Cas-mediated chromosome engineering for crop improvement and synthetic biology. Nat. Plants 7, 566–573 (2021).

    Article  Google Scholar 

  3. Huang, K. & Rieseberg, L. H. Frequency, origins, and evolutionary role of chromosomal inversions in plants. Front. Plant Sci. 11, 296 (2020).

    Article  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Durr, J., Papareddy, R., Nakajima, K. & Gutierrez-Marcos, J. Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9. Sci. Rep. 8, 4443 (2018).

    Article  Google Scholar 

  8. Wu, R. et al. An efficient CRISPR vector toolbox for engineering large deletions in Arabidopsis thaliana. Plant Methods 14, 65 (2018).

    Article  Google Scholar 

  9. Ordon, J. et al. Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J. 89, 155–168 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. Miki, D., Zhang, W., Zeng, W., Feng, Z. & Zhu, J.-K. CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation. Nat. Commun. 9, 1967 (2018).

    Article  Google Scholar 

  14. Wolter, F. & Puchta, H. In planta gene targeting can be enhanced by the use of CRISPR/Cas12a. Plant J. 100, 1083–1094 (2019).

    Article  CAS  Google Scholar 

  15. Merker, L., Schindele, P. & Puchta, H. Using CRISPR/ttLbCas12a for in planta gene targeting in A. thaliana. Curr. Protoc. Plant Biol. 5, e20117 (2020).

    Article  CAS  Google Scholar 

  16. Veillet, F. et al. Transgene-free genome editing in tomato and potato plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor. Int. J. Mol. Sci. 20, https://doi.org/10.3390/ijms20020402 (2019).

  17. Xu, R. et al. Gene targeting using the Agrobacterium tumefaciens–mediated CRISPR–Cas system in rice. Rice 7, 5 (2014).

    Article  Google Scholar 

  18. Char, S. N. et al. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnol. J. 15, 257–268 (2017).

    Article  CAS  Google Scholar 

  19. Zhang, Z. et al. Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing. Plant Biotechnol. J. 17, 1623–1635 (2019).

    Article  CAS  Google Scholar 

  20. Gasparis, S. et al. A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (Hordeum vulgare L.). Plant Methods 14, 111 (2018).

    Article  CAS  Google Scholar 

  21. Huang, T.-K. & Puchta, H. Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering. Transgenic Res. 30, 529–549 (2021).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).

    Article  Google Scholar 

  24. Altpeter, F. et al. Advancing crop transformation in the era of genome editing. Plant Cell 28, 1510–1520 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lowe, K. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28, 1998–2015 (2016).

    Article  CAS  Google Scholar 

  26. Merker, L., Schindele, P., Huang, T.-K., Wolter, F. & Puchta, H. Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a. Plant Biotechnol. J. 18, 2382–2384 (2020).

    Article  CAS  Google Scholar 

  27. Schindele, P., Wolter, F. & Puchta, H. CRISPR guide RNA design guidelines for efficient genome editing. Methods Mol. Biol. 2166, 331–342 (2020).

    Article  CAS  Google Scholar 

  28. Steinert, J., Schmidt, C. & Puchta, H. Use of the Cas9 orthologs from Streptococcus thermophilus and Staphylococcus aureus for non-homologous end-joining mediated site-specific mutagenesis in Arabidopsis thaliana. Methods Mol. Biol. 1669, 365–376 (2017).

    Article  CAS  Google Scholar 

  29. Alonso José, M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).

    Article  Google Scholar 

  30. Graham, T. G. W., Carney, S. M., Walter, J. C. & Loparo, J. J. A single XLF dimer bridges DNA ends during nonhomologous end joining. Nat. Struct. Mol. Biol. 25, 877–884 (2018).

    Article  CAS  Google Scholar 

  31. Kazda, A. et al. Chromosome end protection by blunt-ended telomeres. Genes Dev. 26, 1703–1713 (2012).

    Article  CAS  Google Scholar 

  32. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  33. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  34. Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168–e168 (2014).

    Article  Google Scholar 

  35. Oberpichler, I. et al. Light affects motility and infectivity of Agrobacterium tumefaciens. Environ. Microbiol. 10, 2020–2029 (2008).

    Article  CAS  Google Scholar 

  36. Salomon, S. & Puchta, H. Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J. 17, 6086–6095 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the European Research Council (Advanced grant ERC-2016-AdG_741306 CRISBREED).

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Authors and Affiliations

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

    Michelle Rönspies, Patrick Schindele, Rebecca Wetzel & Holger Puchta

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  1. Michelle Rönspies
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  2. Patrick Schindele
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  4. Holger Puchta
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Contributions

All authors wrote the manuscript. M.R., P.S. and R.W. designed and created the figures.

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Correspondence to Holger Puchta.

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Nature Protocols thanks Goetz Hensel, Seiichi Toki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Beying, N. et al. Nat. Plants 6, 638–645 (2020): https://doi.org/10.1038/s41477-020-0663-x

Schmidt, C. et al. Nat. Commun. 11, 4418 (2020): https://doi.org/10.1038/s41467-020-18277-z

Schmidt, C. et al. Plant J. 98, 577–589 (2019): https://doi.org/10.1111/tpj.14322

Supplementary information

Supplementary Data 1

Raw data from thermal gradient simplex ddPCR (Fig. 4)

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Rönspies, M., Schindele, P., Wetzel, R. et al. CRISPR–Cas9-mediated chromosome engineering in Arabidopsis thaliana. Nat Protoc 17, 1332–1358 (2022). https://doi.org/10.1038/s41596-022-00686-7

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