Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis

Autoři: Yuxin Cheng aff001;  Na Zhang aff001;  Saddam Hussain aff001;  Sajjad Ahmed aff001;  Wenting Yang aff001;  Shucai Wang aff001
Působiště autorů: Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun, Jilin, China aff001;  College of Life Science, Linyi University, Linyi, Shandong, China aff002
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0218583


The CRISPR/Cas9 genome editing technique has been widely used to generate transgene-free mutants in different plant species. Several different methods including fluorescence marker-assisted visual screen of transgene-free mutants and programmed self-elimination of CRISPR/Cas9 construct have been used to increase the efficiency of genome edited transgene-free mutant isolation, but the overall time length required to obtain transgene-free mutants has remained unchanged in these methods. We report here a method for fast generation and easy identification of transgene-free mutants in Arabidopsis. By generating and using a single FT expression cassette-containing CRISPR/Cas9 construct, we targeted two sites of the AITR1 gene. We obtained many early bolting plants in T1 generation, and found that about two thirds of these plants have detectable mutations. We then analyzed T2 generations of two representative lines of genome edited early bolting T1 plants, and identified plants without early bolting phenotype, i.e., transgene-free plants, for both lines. Further more, aitr1 homozygous mutants were successful obtained for both lines from these transgene-free plants. Taken together, these results suggest that the method described here enables fast generation, and at the mean time, easy identification of transgene-free mutants in plants.

Klíčová slova:

Arabidopsis thaliana – CRISPR – Flowering plants – Genetically modified plants – Phenotypes – Plant genomics – Polymerase chain reaction – Seeds


1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337: 816–821. doi: 10.1126/science.1225829 22745249

2. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819–823. doi: 10.1126/science.1231143 23287718

3. Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013; 31: 688–691. doi: 10.1038/nbt.2654 23929339

4. Nekrasov V, Staskawicz B, Weigel D, Jones JD. Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013; 31: 691–693. doi: 10.1038/nbt.2655 23929340

5. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013; 31: 686–688. doi: 10.1038/nbt.2650 23929338

6. Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 2015; 8: 1274–1284. doi: 10.1016/j.molp.2015.04.007 25917172

7. Gao X, Chen J, Dai X, Zhang D, Zhao Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by RISPR/Cas9-mediated genome editing. Plant Physiol. 2016; 171: 1794–1800. doi: 10.1104/pp.16.00663 27208253

8. Lu HP, Liu SM, Xu SL, Chen WY, Zhou X, Tan YY, et al. CRISPR-S: an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants. Plant Biotechnol J. 2017; 15: 1371–1373. doi: 10.1111/pbi.12788 28688132

9. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol. 2017; 35: 441–443. doi: 10.1038/nbt.3833 28346401

10. He Y, Zhu M, Wang L, Wu J, Wang Q, Wang R, et al. Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants. Mol Plant 2018; 11: 1210–1213. doi: 10.1016/j.molp.2018.05.005 29857174

11. Zsögön A, Čermák T, Naves ER, Notini MM, Edel KH, Weinl S, et al. De novo domestication of wild tomato using genome editing. Nat Biotechnol. 2018; 36: 1211–1216.

12. Chen K, Wang Y, Zhang R, Zhang H, Gao C. CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. Annu Rev Plant Biol. 2019; 70: 667–697. doi: 10.1146/annurev-arplant-050718-100049 30835493

13. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 2018; 556: 57–63. doi: 10.1038/nature26155 29512652

14. Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 2018; 361: 1259–1262. doi: 10.1126/science.aas9129 30166441

15. Jung C, Muller AE. Flowering time control and applications in plant breeding. Trends Plant Sci. 2009; 14: 63–573.

16. Boss PK, Bastow RM, Mylne JS, Dean C. Multiple pathways in the decision to flower: enabling, promoting, and resetting. Plant Cell 2004; 16: S18–S31. doi: 10.1105/tpc.015958 15037730

17. Yant L, Mathieu J, Schmid M. Just say no: floral repressors help Arabidopsis bide the time. Curr Opin Plant Biol. 2009; 12: 580–586. doi: 10.1016/j.pbi.2009.07.006 19695946

18. Wahl V, Ponnu J, Schlereth A, Arrivault S, Langenecker T, Franke A, et al. Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana. Science 2013; 339: 704–707. doi: 10.1126/science.1230406 23393265

19. Gendall AR, Levy YY, Wilson A, Dean C. The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell 2001; 107: 525–535. doi: 10.1016/s0092-8674(01)00573-6 11719192

20. Ratcliffe OJ, Kumimoto RW, Wong BJ, Riechmann JL. Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold. Plant Cell 2003; 15: 1159–1169. doi: 10.1105/tpc.009506 12724541

21. Mockler TC, Yu XH, Shalitin D, Parikh D, Michael TP, Liou J, et al. Regulation of flowering time in Arabidopsis by K homology domain proteins. Proc Natl Acad Sci USA 2004; 101: 12759–12764. doi: 10.1073/pnas.0404552101 15310842

22. Tamada Y, Yun JY, Woo SC. Amasino R.M ARABIDOPSIS TRITHORAX-RELATED7 is required for methylation of lysine 4 of histone H3 and for transcriptional activation of FLOWERING LOCUS C. Plant Cell 2009; 21: 3257–3269. doi: 10.1105/tpc.109.070060 19855050

23. Wang JW, Czech B, Weigel D. miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell 2009; 138: 738–749. doi: 10.1016/j.cell.2009.06.014 19703399

24. Jung JH, Seo PJ, Ahn JH, Park CM. Arabidopsis RNA-binding protein FCA regulates microRNA172 processing in thermosensory flowering. J Biol Chem. 2012; 287: 16007–16016. doi: 10.1074/jbc.M111.337485 22431732

25. Tamaki S, Matsuo S, Wong HL. Yokoi S. Shimamoto K. Hd3a protein is a mobile flowering signal in rice. Science 2007; 316: 1033–1036. doi: 10.1126/science.1141753 17446351

26. Kong F, Liu B. Xia Z, Sato S, Kim BM, Watanabe S, et al. Two coordinately regulated homologs of FLOWERING LOCUS T are involved in the control of photoperiodic flowering in soybean. Plant Physiol. 2010; 154: 1220–1231. doi: 10.1104/pp.110.160796 20864544

27. Putterill J, Varkonyi-Gasic E. FT and florigen long-distance flowering control in plants. Curr Opin Plant Biol. 2016; 33: 77–82. doi: 10.1016/j.pbi.2016.06.008 27348248

28. Wickland DP, Hanzawa Y. The FLOWERING LOCUS T/TERMINAL FLOWER 1 gene family: functional evolution and molecular mechanisms. Mol Plant 2015; 8: 983–997. doi: 10.1016/j.molp.2015.01.007 25598141

29. Bull SE, Seung D, Chanez C, Mehta D, Kuon JE, Truernit E, et al. Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch. Sci Adv. 2018; 4: eaat6086. doi: 10.1126/sciadv.aat6086 30191180

30. Tian H, Chen S, Yang W, Wang T, Zheng K, Wang Y, et al. A novel family of transcription factors conserved in angiosperms is required for ABA signalling. Plant Cell Environ. 2017; 40: 2958–2971. doi: 10.1111/pce.13058 28857190

31. Tian H, Guo H. Dai X. Cheng Y. Zheng K. Wang X. et al. An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in Arabidopsis. Scic Rep. 2015; 5: 17587. doi: 10.1038/srep17587 26625868

32. Dai X, Zhou L, Zhang W, Cai L, Guo H. Tian H. et al. A single amino acid substitution in the R3 domain of GLABRA1 leads to inhibition of trichome formation in Arabidopsis without affecting its interaction with GLABRA3. Plant Cell Environ. 2016; 39: 897–907. doi: 10.1111/pce.12695 26667588

33. Wang Z, Xing H, Dong L, Zhang H, Han C, Wang X, 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. 2015; 16: 144. doi: 10.1186/s13059-015-0715-0 26193878

34. Wang S, Tiwari SB, Hagen G, Guilfoyle TJ. AUXIN RESPONSE FACTOR7 restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts. Plant Cell 2005; 17: 1979–1993. doi: 10.1105/tpc.105.031096 15923351

35. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16: 735–743. doi: 10.1046/j.1365-313x.1998.00343.x 10069079

36. Guo H, Zhang W, Tian H, Zheng K, Dai X, Liu S, et al. An auxin responsive CLE gene regulates shoot apical meristem development in Arabidopsis. Front Plant Sci. 2015; 6: 295. doi: 10.3389/fpls.2015.00295 25983737

37. Sun H, Jia Z, Cao D, Jiang B, Wu C, Hou W, et al. GmFT2a, a soybean homolog of FLOWERING LOCUS T, is involved in flowering transition and maintenance. PLoS One 2011; 6: e29238. doi: 10.1371/journal.pone.0029238 22195028

38. Nan H, Cao D, Zhang D, Li Y, Lu S, Tang L, et al. GmFT2a and GmFT5a redundantly and differentially regulate flowering through interaction with and upregulation of the bZIP transcription factor GmFDL19 in soybean. PLoS One 2014; 9: e97669. doi: 10.1371/journal.pone.0097669 24845624

39. Putterill J, Zhang L, Yeoh C, Balcerowicz M, Jaudal M, Varkonyi Gasic E. FT genes and regulation of flowering in the legume Medicago truncatula. Funct Plant Biol. 2013; 40: 1199–1207.

40. Yamagishi N, Kishigami R, Yoshikawa N. Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant-breeding technique with no transmission of genetic modification to the next generation. Plant Biotechnol J. 2014; 12: 60–68. doi: 10.1111/pbi.12116 23998891

41. Yamagishi N, Li C, Yoshikawa N. Promotion of flowering by Apple latent spherical virus vector and virus elimination at high temperature allow accelerated breeding of apple and pear. Front Plant Sci. 2016; 7: 171. doi: 10.3389/fpls.2016.00171 26941750

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