DOI QR코드

DOI QR Code

Genome-wide in-locus epitope tagging of Arabidopsis proteins using prime editors

  • Cheljong Hong (Department of Chemistry, Seoul National University) ;
  • Jun Hee Han (Department of Chemistry, Hanyang University) ;
  • Gue-Ho Hwang (Research Center of Genomic Medicine Institute, Seoul National University College of Medicine) ;
  • Sangsu Bae (Research Center of Genomic Medicine Institute, Seoul National University College of Medicine) ;
  • Pil Joon Seo (Department of Chemistry, Seoul National University)
  • Received : 2023.04.11
  • Accepted : 2023.06.19
  • Published : 2024.01.31

Abstract

Prime editors (PEs), which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusion proteins programmed with prime editing guide RNAs (pegRNAs), can not only edit bases but also install transversions, insertions, or deletions without both donor DNA and double-strand breaks at the target DNA. As the demand for in-locus tagging is increasing, to reflect gene expression dynamics influenced by endogenous genomic contexts, we demonstrated that PEs can be used to introduce the hemagglutinin (HA) epitope tag to a target gene locus, enabling molecular and biochemical studies using in-locus tagged plants. To promote genome-wide in-locus tagging, we also implemented a publicly available database that designs pegRNAs for in-locus tagging of all the Arabidopsis genes.

Keywords

Acknowledgement

Analysis of sequencing data was mostly carried out using the computing server at the Genomic Medicine Institute Research Service Center. This research was supported by grants from the National Research Foundation of Korea (NRF) no. 2021R1AC3012908 to S.B. and no. NRF-2022R1A2B5B02001266 to P.J.S. This work was also supported by a grant from the New breeding technologies development Program (PJ01653002 to P.J.S.), Rural Development Administration.

References

  1. Anzalone AV, Randolph PB, Davis JR et al (2019) Search-nd-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 https://doi.org/10.1038/s41586-019-1711-4
  2. Butt H, Rao GS, Sedeek K, Aman R, Kamel R and Mahfouz M (2020) Engineering herbicide resistance via prime editing in rice. Plant Biotechnol J 18, 2370-2372 https://doi.org/10.1111/pbi.13399
  3. Hua K, Jiang Y, Tao X and Zhu JK (2020) Precision genome engineering in rice using prime editing system. Plant Biotechnol J 18, 2167-2169 https://doi.org/10.1111/pbi.13395
  4. Jiang YY, Chai YP, Lu MH et al (2020) Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biol 21, 257
  5. Lu YM, Tian YF, Shen RD et al (2021) Precise genome modification in tomato using an improved prime editing system. Plant Biotechnol J 19, 415-417 https://doi.org/10.1111/pbi.13497
  6. Li HY, Li JY, Chen JL, Yan L and Xia LQ (2020) Precise modifications of both exogenous and endogenous genes in rice by prime editing. Mol Plant 13, 671-674 https://doi.org/10.1016/j.molp.2020.03.011
  7. Lin QP, Zong Y, Xue CX et al (2020) Prime genome editing in rice and wheat. Nat Biotechnol 38, 582-585 https://doi.org/10.1038/s41587-020-0455-x
  8. Tang X, Sretenovic S, Ren QR et al (2020) Plant prime editors enable precise gene editing in rice cells. Mol Plant 13, 667-670 https://doi.org/10.1016/j.molp.2020.03.010
  9. Xu RF, Li J, Liu XS, Shan TF, Qin RY and Wei PC (2020) Development of plant prime-editing systems for precise genome editing. Plant Commun 1, 100043
  10. Xue C, Qiu F, Wang Y et al (2023) Tuning plant phenotypes by precise, graded downregulation of gene expression. Nat Biotechnol doi: 10.1038/s41587-023-01707-w
  11. Lackner DH, Carre A, Guzzardo PM et al (2015) A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat Commun 6, 10237
  12. Meurer M, Duan YQ, Sass E et al (2018) Genome-wide C-SWAT library for high-throughput yeast genome tagging. Nat Methods 15, 598-600 https://doi.org/10.1038/s41592-018-0045-8
  13. Jiang J, Yan M, Li DS and Li JS (2019) Genome tagging project: tag every protein in mice through 'artificial spermatids'. Natl Sci Rev 6, 394-396 https://doi.org/10.1093/nsr/nwy136
  14. Lu YM, Ronald PC, Han B, Li JY and Zhu JK (2020) Rice protein tagging project: a call for international collaborations on genome-wide in-locus tagging of rice proteins. Mol Plant 13, 1663-1665 https://doi.org/10.1016/j.molp.2020.11.006
  15. Nishimasu H, Shi X, Ishiguro S et al (2018) Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259-1262 https://doi.org/10.1126/science.aas9129
  16. Perroud PF, Guyon-Debast A, Veillet F et al (2022) Prime editing in the model plant physcomitrium patens and its potential in the tetraploid potato. Plant Sci 316, 111162
  17. Wang L, Kaya HB, Zhang N et al (2021) Spelling changes and fluorescent tagging with prime editing vectors for plants. Front Genome Ed 3, 617553
  18. Xu W, Zhang CW, Yang YX et al (2020) Versatile nucleotides substitution in plant using an improved prime editing system. Mol Plant 13, 675-678 https://doi.org/10.1016/j.molp.2020.03.012
  19. Ma XN, Zhang XY, Liu HM and Li ZH (2020) Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9. Nat Plants 6, 773-779 https://doi.org/10.1038/s41477-020-0704-5
  20. Jeong YY, Lee HY, Kim SW, Noh YS and Seo PJ (2021) Optimization of protoplast regeneration in the model plant Arabidopsis thaliana. Plant Methods 17, 21
  21. Wang ZY and Tobin EM (1998) Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 93, 1207-1217 https://doi.org/10.1016/S0092-8674(00)81464-6
  22. Park J, Bae S and Kim JS (2015) Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31, 4014-4016 https://doi.org/10.1093/bioinformatics/btv537
  23. Hwang GH, Jeong YK, Habib O et al (2021) PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res 49, W499-W504 https://doi.org/10.1093/nar/gkab319
  24. Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743 https://doi.org/10.1046/j.1365-313x.1998.00343.x