Advertisement

CRISPR/Cas9-mediated homologous recombination in tobacco

  • Ayumi Hirohata
  • Izumi Sato
  • Kimihiko Kaino
  • Yuji Iwata
  • Nozomu Koizumi
  • Kei-ichiro Mishiba
Original Article

Abstract

Key message

Co-transformation of multiple T-DNA in a binary vector enabled CRISPR/Cas9-mediated HR in tobacco. HR occurred in a limited region around the gRNA target site.

Abstract

In this study, CRISPR/Cas9-mediated homologous recombination (HR) in tobacco (Nicotiana tabacum L. ‘SR-1’) was achieved using binary vectors comprising two (T1–T2) or three (T1–T2–T3) independent T-DNA regions. For HR donor with the tobacco acetolactate synthase gene, SuRB, T-DNA1 contained ΔSuRBW568L, which lacked the N-terminus region of SuRB and was created by three nucleotide substitutions (ATG to GCT; W568L), leading to herbicide chlorsulfuron (Cs) resistance, flanked by the hygromycin (Hm)-resistant gene. T-DNA2 consisted of the hSpCas9 gene and two gRNA inserts targeting SuRB and An2. For the 2nd HR donor with the tobacco An2 gene encoding a MYB transcription factor involved in anthocyanin biosynthesis, T-DNA3 had a 35S promoter-driven An2 gene lacking the 3rd exon resulting in anthocyanin accumulation after successful HR. After selecting for Hm and Cs resistance from among the 7462 Agrobacterium-inoculated explants, 77 independent lines were obtained. Among them, the ATG to GCT substitution of endogenous SuRB was detected in eight T1–T2-derived lines and two T1–T2–T3-derived lines. Of these mutations, four T1–T2-derived lines were bi-allelic. All the HR events occurred across the endogenous SuRB and 5′ homology arm of the randomly integrated T-DNA1. HR of the SuRB paralog, SuRA, was also found in one of the T1–T2-derived lines. Sequence analysis of its SuRA-targeted region indicated that the HR occurred in a limited (< 153 bp) region around the gRNA target site. Even though some T1–T2–T3-derived lines introduced three different T-DNAs and modified the An2 gRNA target site, no signs of HR in the endogenous An2 could be observed.

Keywords

Homologous recombination CRISPR/Cas9 T-DNA Tobacco 

Abbreviations

CRISPR

Clustered regularly interspaced short palindromic repeats

DSBs

Double-strand breaks. HMA, heteroduplex mobility assay

GT

Gene targeting

HDR

Homology-directed repair

HR

Homologous recombination

NHEJ

Nonhomologous end-joining

Notes

Acknowledgements

The authors would like to thank Dr. Roger Hellens (Queensland University of Technology), Dr. Philip Mullineaux (University of Essex) and Dr. Mark Smedley (John Innes Centre) for the pGreen vector system, Dr. Feng Zhang (Massachusetts Institute of Technology) for the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, Dr. Nam-Hai Chua (Rockefeller University) for the pER8 vector, and Dr. Hiroaki Ichikawa (NIAS, Japan) for the pSMAB704 vector. We also thank Editage (http://www.editage.jp) for English language editing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2018_2320_MOESM1_ESM.doc (100 kb)
Supplementary material 1 (DOC 100 KB)
299_2018_2320_MOESM2_ESM.pdf (1.7 mb)
Supplementary material 2 (PDF 1705 KB)

References

  1. Beetham PR, Kipp PB, Sawycky XL, Arntzen CJ, May GD (1999) A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proc Natl Acad Sci USA 96:8774–8778CrossRefPubMedGoogle Scholar
  2. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52CrossRefPubMedGoogle Scholar
  3. Čermák T, Baltes NJ, Čegan R, Zhang Y, Voytas DF (2015) High-frequency, precise modification of the tomato genome. Genome Biol 16:232CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 1:581–585CrossRefPubMedGoogle Scholar
  5. 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 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cortleven A, Remans T, Brenner WG, Valcke R (2009) Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum. Photosynth Res 102:21–29CrossRefPubMedPubMedCentralGoogle Scholar
  7. Coruzzi G, Broglie R, Edwards C, Chua NH (1984) Tissue-specific and light-regulated expression of a pea nuclear gene encoding the small subunit of ribulose-l,5-bisphosphate carboxylase. EMBO J 3:1671–1679PubMedPubMedCentralGoogle Scholar
  8. Delwart EL, Shpaer EG, Louwagie J, McCutchan FE, Grez M, Rübsamen-Waigmann H, Mullins JI (1993) Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes. Science 19:1261–1275Google Scholar
  9. Dong X, Stothard P, Forsythe IJ, Wishart DS (2004) PlasMapper: a web server for drawing and auto-annotating plasmid maps. Nucl Acids Res 32:W660–W664CrossRefPubMedGoogle Scholar
  10. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349CrossRefPubMedPubMedCentralGoogle Scholar
  11. Endo M, Osakabe K, Ichikawa H, Toki S (2006) Molecular characterization of true and ectopic gene targeting events at the acetolactate synthase gene in Arabidopsis. Plant Cell Physiol 47:372–379CrossRefPubMedGoogle Scholar
  12. Endo M, Osakabe K, Ono K, Handa H, Shimizu T, Toki S (2007) Molecular breeding of a novel herbicide-tolerant rice by gene targeting. Plant J 52:157–166CrossRefPubMedGoogle Scholar
  13. Endo M, Mikami M, Toki S (2015) Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol 56:41–47CrossRefPubMedGoogle Scholar
  14. Endo M, Mikami M, Toki S (2016) Biallelic gene targeting in rice. Plant Physiol 170:667–677CrossRefPubMedGoogle Scholar
  15. Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu JK (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23:1229–1232CrossRefPubMedPubMedCentralGoogle Scholar
  16. Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu JK (2014) Multi-generation analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637CrossRefPubMedGoogle Scholar
  17. Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, Wu Y, Zhao P, Xia Q (2015) CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol 87:99–110CrossRefPubMedGoogle Scholar
  18. Hanin M, Volrath S, Bogucki A, Briker M, Ward E, Paszkowski J (2001) Gene targeting in Arabidopsis. Plant J 28:671–677CrossRefPubMedGoogle Scholar
  19. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832CrossRefPubMedGoogle Scholar
  20. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282CrossRefPubMedGoogle Scholar
  21. Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucl Acids Res 16:9877CrossRefPubMedGoogle Scholar
  22. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301CrossRefPubMedPubMedCentralGoogle Scholar
  23. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  24. Igasaki T, Ishida Y, Mohri T, Ichikawa H, Shinohara K (2002) Transformation of Populus alba and direct selection of transformants with the herbicide bialaphos. Bull FFPRI 1:235–240Google Scholar
  25. Ishige F, Takaichi M, Foster R, Chua NH, Oeda K (1999) A G-box motif (GCCACGTGCC) tetramer confers high-level constitutive expression in dicot and monocot plants. Plant J 18:443–448CrossRefGoogle Scholar
  26. Jiang W, Yang B, Weeks DP (2014) Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations. PLOS ONE 9:e99225CrossRefPubMedPubMedCentralGoogle Scholar
  27. 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 337:816–821CrossRefPubMedGoogle Scholar
  28. Kochevenko A, Willmitzer L (2003) Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate syntase gene. Plant Physiol 132:174–184CrossRefPubMedPubMedCentralGoogle Scholar
  29. Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174CrossRefPubMedGoogle Scholar
  30. Lee KY, Townsend J, Tepperman J, Black M, Chui CF, Mazur B, Dunsmuir P, Bedbrook J (1988) The molecular basis of sulfonylurea herbicide resistance in tobacco. EMBO J 7:1241–1248PubMedPubMedCentralGoogle Scholar
  31. Lee KY, Lund P, Lowe K, Dunsmuir P (1990) Homologous recombination in plant cells after Agrobacterium-mediated transformation. Plant Cell 2:415–425CrossRefPubMedPubMedCentralGoogle Scholar
  32. 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 31:688–691CrossRefPubMedPubMedCentralGoogle Scholar
  33. Li Z, Liu ZB, Xing A, Moon BP, Koellhoffer JP, Huang L, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960–970CrossRefPubMedPubMedCentralGoogle Scholar
  34. Ma X, Zhu Q, Chen Y, Liu YG (2017) CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9:961–974CrossRefGoogle Scholar
  35. Mercx S, Tollet J, Magy B, Navarre C, Boutry M (2016) Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells. Front Plant Sci 7:40CrossRefPubMedPubMedCentralGoogle Scholar
  36. Mercx S, Smargiasso N, Chaumont F, Pauw ED, Boutry M, Navarre C (2017) Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 cells by a multiplex CRISPR/Cas9 strategy results in glycoproteins without plant-specific glycans. Front Plant Sci 8:403CrossRefPubMedPubMedCentralGoogle Scholar
  37. Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23:1233–1236CrossRefPubMedPubMedCentralGoogle Scholar
  38. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  39. Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693CrossRefPubMedGoogle Scholar
  40. Okuzaki A, Toriyama K (2004) Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice. Plant Cell Rep 22:509–512CrossRefPubMedGoogle Scholar
  41. Pattanaik S, Kong Q, Zaitlin D, Werkman JR, Xie CH, Patra B, Yuan L (2010) Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231:1061–1076CrossRefPubMedGoogle Scholar
  42. Saika H, Mori A, Endo M, Osakabe K, Toki S (2015) Rapid evaluation of the frequency of gene targeting in rice via a convenient positive-negative selection method. Plant Biotechnol 32:169–173CrossRefGoogle Scholar
  43. Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355CrossRefPubMedPubMedCentralGoogle Scholar
  44. Schiml S, Fauser F, Puchta H (2014) The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J 80:1139–1150CrossRefPubMedGoogle Scholar
  45. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688CrossRefPubMedGoogle Scholar
  46. Steinert J, Schiml S, Puchta H (2016) Homology-based double-strand break-induced genome engineering in plants. Plant Cell Rep 35:1429–1438CrossRefPubMedGoogle Scholar
  47. Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L (2016) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol Plant 9:628–631CrossRefPubMedGoogle Scholar
  48. Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169:931–945CrossRefPubMedPubMedCentralGoogle Scholar
  49. Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459:442–445CrossRefPubMedPubMedCentralGoogle Scholar
  50. Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951CrossRefPubMedGoogle Scholar
  51. Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–3575CrossRefPubMedGoogle Scholar
  52. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161:20–27CrossRefPubMedGoogle Scholar
  54. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu JK (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807CrossRefPubMedGoogle Scholar
  55. Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zhao Y, Zhang C, Liu W, Gao W, Liu C, Song G, Li WX, Mao L, Chen B, Xu Y, Li X, Xie C (2016) An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Sci Rep 6:23890CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhu T, Peterson DJ, Tagliani L, St. Clair G, Baszczynski CL, Bowen B (1999) Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 96:8768–8773CrossRefPubMedGoogle Scholar
  58. Zuo J, Niu QW, Chua NH (2001) An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Graduate School of Life and Environmental SciencesOsaka Prefecture UniversitySakaiJapan

Personalised recommendations