Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 129, Issue 1, pp 153–160 | Cite as

Dual-targeting by CRISPR/Cas9 for precise excision of transgenes from rice genome

  • Vibha Srivastava
  • Jamie L. Underwood
  • Shan Zhao
Original Article


The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has emerged as the robust gene editing tool that functions through the double-stranded break repair process leading to targeted mutagenesis in higher genomes. CRISPR/Cas9 has been simplified to a two component system consisting of a single guide RNA (gRNA) that binds Cas9 to target genomic sites in sequence-dependent manner. This RNA-guided nuclease system has mostly been applied for inducing point mutations or short insertion-deletions at one or multiple loci. The present study addressed the utility of this system for excising marker genes from plant genomes, an application highly relevant for developing marker-free transgenic plants. A transgenic rice line expressing β-glucuronidase (GUS) gene was transformed by Agrobacterium or gene gun with a construct expressing Cas9 and two gRNAs to target each end of 1.6 kb GUS gene. Molecular analysis of the transformed lines detected excision at low frequency in the callus lines, but at significantly higher frequency in plant lines, indicating robust efficiency of Cas9:gRNA in regenerated plants. Bi-allelic excisions were observed in plants derived from three independent events, allowing recovery of homozygous excision lines in the first generation (T0). Notably, the excision in different plant lines was formed by precise cut and ligation of the two blunt ends without mutation at or around the excision site. Since the goal of marker-removal technologies is to precisely excise a defined piece of DNA without introducing mutations in the adjacent sequences, Cas9:gRNA system could be an effective tool for producing marker-free plants.


CRISPR/Cas9 gRNA Multiplex gene editing Marker excision Rice transformation 



The vectors pRGEB32, pRGE32 and pGTR were donated by Yinong Yang and obtained from This project is supported by the Arkansas Division of Agriculture.

Author contributions

JLU developed vectors and performed a part of rice transformations. SZ did most of the rice transformations and PCR analysis. VS analyzed the data and wrote the manuscript.


  1. Antunes MS, Smith JJ, Jantz D, Medford JI (2012) Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease. BMC Biotechnol 12:86. doi: 10.1186/1472-6750-12-86 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Baltes NJ, Voytas DF (2015) Enabling plant synthetic biology through genome engineering. Trends Biotechnol 33:120–131. doi: 10.1016/j.tibtech.2014.11.008 CrossRefPubMedGoogle Scholar
  3. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52. doi: 10.1016/j.biotechadv.2014.12.006 CrossRefPubMedGoogle Scholar
  4. Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297. doi: 10.1104/pp.114.247577 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chen K, Gao C (2014) Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep 33:575–583. doi: 10.1007/s00299-013-1539-6 CrossRefPubMedGoogle Scholar
  6. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi: 10.1126/science.1231143 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Feng ZY, Mao YF, Xu NF, Zhang BT, Wei PL, Wang Z, Zhang ZJ, Yang DL, Yang L, Zeng L, Liu XD, Zhu J-K (2014) Multi-generation analysis reveals the inheritance, specificity and patterns of CRISPR/Cas induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637. doi: 10.1073/pnas.1400822111 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gao X, Chen J, Dai X, Zhang D, Zhao Y (2016) An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol. doi: 10.1104/pp.16.00663 Google Scholar
  9. Gidoni D, Srivastava V, Carmi N (2008) Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants. In Vitro Cell Dev Biol-Plant 44:457–467. doi: 10.1007/s11627-008-9140-3 CrossRefGoogle Scholar
  10. Gilbertson L (2003) Cre-lox recombination: cre-ative tools for plant biotechnology. Trends Biotech 21:550–555. doi: 10.1016/j.tibtech.2003.09.011 CrossRefGoogle Scholar
  11. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  12. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. doi: 10.1126/science.1225829 CrossRefPubMedGoogle Scholar
  13. Khattri A, Nandy S, Srivastava V (2011) Heat-inducible cre–lox system for marker excision in transgenic rice. J Biosci 36:37–42. doi: 10.1007/s12038-011-9010-8 CrossRefPubMedGoogle Scholar
  14. Lemaux PG (2008) Genetically engineered plants and foods: a scientist’s analysis of the issues (Part I). Annu Rev. Plant Biol 59:771–812. doi: 10.1146/annurev.arplant.58.032806.103840 CrossRefGoogle Scholar
  15. Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691. doi: 10.1038/nbt.2654 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Luo K, Duan H, Zhao D, Zheng X, Deng X, Chen Y, Stewart CN Jr, McAvoy R, Wu Y, Jiang X, He A, Pei Y, Li Y (2007) ‘GM-gene-deletor’: fusedloxP-FRTrecognition sequences dramatically improve efficiency of FLP or Cre recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–374. doi: 10.1111/j.1467-7652.2006.00237.x CrossRefPubMedGoogle Scholar
  17. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. doi: 10.1126/science.1232033 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Nandy S, Srivastava V (2012) Marker-free site-specific gene integration in rice based on the use of two recombination systems. Plant Biotech J. 10:904–912. doi: 10.1111/j.1467-7652.2012.00715.x CrossRefGoogle Scholar
  19. Nandy S, Zhao S, Pathak B, Manoharan M, Srivastava V (2015) Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion. BMC Biotechnol. 15:93 doi: 10.1186/s12896-015-0212-2 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Nishimura A, Aichi I, Matsuoka M (2006) A protocol for Agrobacterium mediated transformation in rice. Nat Protoc 1:2796–2802. doi: 10.1038/nprot.2006.469 CrossRefPubMedGoogle Scholar
  21. Ordon J, Gantner J, Kemna J, Schwalgun L, Reschke M, Streubel J, Boch J, Stuttmann J (2016) Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J. doi: 10.1111/tpj.13319 PubMedGoogle Scholar
  22. Ow DW (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200. doi: 10.1023/A:1013718106742 CrossRefPubMedGoogle Scholar
  23. Petolino JF, Worden A, Curlee K, Connell J, Strange Moynahan TL, Larsen C, Russell S (2010) Zinc finger nuclease-mediated transgene deletion. Plant Mol Biol 73:617–628. doi: 10.1007/s11103-010-9641-4 CrossRefPubMedGoogle Scholar
  24. Siebert R, Puchta H (2002) Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome. Plant Cell 14:1121–1131. doi: 10.1105/tpc.001727 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Srivastava V, Thomson J (2016) Gene stacking by recombinases. Plant Biotechnol J 14:471–482. doi: 10.1111/pbi.12459 CrossRefPubMedGoogle Scholar
  26. Srivastava V, Akbudak MA, Nandy S (2011) Marker-free plant transformation. In: Dan Y, Ow DW (eds) Historical technology developments in plant transformation. Bentham eBooks, pp 108–122. doi: 10.2174/97816080524861110101
  27. Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976. doi: 10.1111/j.1365-313X.2006.02836.x CrossRefPubMedGoogle Scholar
  28. Wang Y, Yau YY, Perkins-Balding D, Thomson JG (2011) Recombinase technology: applications and possibilities. Plant Cell Rep. 30:267–285. doi: 10.1007/s00299-010-0938-1 CrossRefPubMedGoogle Scholar
  29. Weeks DP, Spalding MH, Yang B (2016) Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnol J 14:483–495. doi: 10.1111/pbi.12448 CrossRefPubMedGoogle Scholar
  30. Wright AV, Nuñez JK, Doudna JA (2016) Biology and applications of CRISPR Systems: Harnessing nature’s toolbox for genome engineering. Cell 164:29–44. doi: 10.1016/j.cell.2015.12.035 CrossRefPubMedGoogle Scholar
  31. Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–3575. doi: 10.1073/pnas.1420294112 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Xu RF, Li H, Qin RY, Li J, Qiu CH, Yang YC, Ma H, Li L, Wei PC, Yang JB (2015) Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep. 5:11491. doi: 10.1038/srep11491.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Yau YY, Stewart CN Jr (2013) Less is more: strategies to remove marker genes from transgenic plants. BMC Biotechnol 13:36. doi: 10.1186/1472-6750-13-36 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Xu N, Zhu JK (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807. doi: 10.1111/pbi.12200 CrossRefPubMedGoogle Scholar
  35. Zhang Z, Mao Y, Ha S, Liu W, Botella JR, Zhu JK (2016) A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Rep 35:1519–1533. doi: 10.1007/s00299-015-1900-z CrossRefPubMedGoogle Scholar
  36. Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42:10903–10914. doi: 10.1093/nar/gku806 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Department of Crop, Soil & Environmental SciencesUniversity of ArkansasFayettevilleUSA
  2. 2.Department of HorticultureUniversity of ArkansasFayettevilleUSA

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