Skip to main content

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells

Abstract

Key message

A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells.

Abstract

Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. ‘Bright Yellow-2’ (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K (2015) Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol 16:87

    PubMed  PubMed Central  Google Scholar 

  2. Almofti MR, Harashima H, Shinohara Y, Almofti A, Li W, Kiwada H (2003) Lipoplex size determines lipofection efficiency with or without serum. Mol Membr Biol 20:35–43

    CAS  PubMed  Google Scholar 

  3. Alsaiari SK, Patil S, Alyami M, Alamoudi KO, Aleisa FA, Merzaban JS, Li M, Khashab NM (2018) Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. J Am Chem Soc 140:143–146

    CAS  PubMed  Google Scholar 

  4. Altpeter F, Springer NM, Bartley LE, Blechl AE, Brutnell TP, Citovsky V, Conrad LJ, Gelvin SB, Jackson DP, Kausch AP, Lemaux PG, Medford JI, Orozco-Cardenas M, Tricoli DM, Eck JV, Voytas DF, Walbot V, Wang K, Zhang ZJ, Stewart CN Jr (2016) Advancing crop transformation in the era of genome editing. Plant Cell 28:1510–1520

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Andersson M, Turesson H, Olsson N, Falt A-S, Ohlsson P, Gonzalez MN, Samuelsson M, Hofvander P (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant 164:378–384

    CAS  PubMed  Google Scholar 

  6. Araki M, Ishii T (2015) Towards social acceptance of plant breeding by genome editing. Trends Plant Sci 20:145–149

    CAS  PubMed  Google Scholar 

  7. Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim J-S, Jin ES, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6:30620

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84

    CAS  PubMed  Google Scholar 

  9. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52

    CAS  Article  Google Scholar 

  10. Buntru M, Vogel S, Spiegel H, Schillberg S (2014) Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnol 14:37

    PubMed  PubMed Central  Google Scholar 

  11. Burger A, Lindsay H, Felker A, Hess C, Anders C, Chiavacci E, Zaugg J, Weber LM, Catena R, Jinek M, Robinson MD, Mosimann C (2016) Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development 143:2025–2037

    CAS  PubMed  Google Scholar 

  12. Centomani I, Sgobba A, D’Addabbo P, Dipierro N, Paradiso A, De Gara L (2015) Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells. Protoplasma 252:1451–1459

    CAS  PubMed  Google Scholar 

  13. Chaverra-Rodriguez D, Macias VM, Hughes GL, Pujhari S, Suzuki Y, Peterson DR, Kim D, McKeand S, Rasgon JL (2018) Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat Commun 9:3008

    PubMed  PubMed Central  Google Scholar 

  14. Chen S, Lee B, Lee AY-F, Modzelewski AJ, He L (2016) Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291:14457–14467

    CAS  PubMed  PubMed Central  Google Scholar 

  15. DeWitt MA, Corn JE, Carroll D (2017) Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121–122:9–15

    PubMed  PubMed Central  Google Scholar 

  16. Feng Z, Zhang B, Ding W, Liu X, Yang D-L, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu J-K (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23:1229–1232

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Frame BR, Zhang H, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE, Zhen S, Schnable PS, Wang K (2000) Production of transgenic maize from bombarded type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol Plant 36:21–29

    Google Scholar 

  18. Gaj T, Gersbach CA, Barbas CF III (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Glass Z, Lee M, Li Y, Xu Q (2018) Engineering the delivery system for CRISPR-based genome editing. Trends Biotechnol 36:173–185

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Han X, Liu Z, Ma Y, Zhang K, Qin L (2017) Cas9 ribonucleoprotein delivery via microfluidic cell-deformation chip for human T-cell genome editing and immunotherapy. Adv Biosys 1:1600007

    Google Scholar 

  21. Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422

    CAS  PubMed  Google Scholar 

  22. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    CAS  Google Scholar 

  23. 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 227:816–821

    Google Scholar 

  24. Kato N, Esaka M (2000) Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts. Planta 210:1018–1022

    CAS  PubMed  Google Scholar 

  25. Kato K, Matsumoto T, Koiwai A, Mizusaki S, Nishida K, Noguchi M, Tamaki E (1972) Liquid suspension culture of tobacco cells. In: Terui G (ed) Fermentation Technology Today. Society of Fermentation Technology of Japan, Osaka, pp 689–695

    Google Scholar 

  26. Kim S, Kim D, Cho SW, Kim J, Kim J-S (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Landry MP, Mitter N (2019) How nanocarriers delivering cargos in plants can change the GMO landscape. Nat Nanotechnol 14:512–514

    CAS  PubMed  Google Scholar 

  28. Lee L-Y, Wu F-H, Hsu C-T, Shen S-C, Yeh H-Y, Liao D-C, Fang M-J, Liu N-T, Yen Y-C, Dokladal L, Sykorova E, Gelvin SB, Lin C-S (2012) Screening a cDNA library for protein-protein interactions directly in planta. Plant Cell 24:1746–1759

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lee K, Conboy M, Park HM, Jiang F, Kim HJ, Dewitt MA, Mackley VA, Chang K, Rao A, Skinner C, Shobha T, Mehdipour M, Liu H, Huang W-C, Lan F, Bray NL, Li S, Corn JE, Kataoka K, Doudna JA, Conboy I, Murthy N (2017) Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 1:889–901

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Lee B, Lee K, Panda S, Gonzales-Rojas R, Chong A, Bugay V, Park HM, Brenner R, Murthy N, Lee HY (2018) Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat Biomed Eng 2:497–507

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53

    CAS  PubMed  Google Scholar 

  32. Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu W, Yuan JS, Stewart CN Jr (2013) Advanced genetic tools for plant biotechnology. Nat Rev Genet 14:781–793

    CAS  PubMed  Google Scholar 

  34. Liu W, Rudis MR, Peng Y, Mazarei M, Millwood RJ, Yang J-P, Xu W, Chesnut JD, Stewart CN Jr (2014a) Synthetic TAL effectors for targeted enhancement of transgene expression in plants. Plant Biotechnol J 12:436–446

    CAS  PubMed  Google Scholar 

  35. Liu W, Mazarei M, Peng Y, Fethe MH, Rudis MR, Lin J, Millwood RJ, Arelli PR, Stewart CN Jr (2014b) Computational discovery of soybean promoter cis-regulatory elements for the construction of soybean cyst nematode inducible synthetic promoters. Plant Biotechnol J 12:1015–1026

    CAS  PubMed  Google Scholar 

  36. Maccarrone M, Dini L, Marzio LD, Giulio AD, Rossi A, Mossa G, Finazzi-Agro A (1992) Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts. Biochem Biophys Res Commun 186:1417–1422

    CAS  PubMed  Google Scholar 

  37. Maccarrone M, Marzio LD, Rossi A, Finazzi-Agro A (1993) Gene transfer to lentil protoplasts by lipofection and electroporation. J Liposome Res 3:707–716

    CAS  Google Scholar 

  38. Malnoy M, Viola R, Jung M-H, Koo O-J, Kim S, Kim J-S, Velasco R, Kanchiswamy CN (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7:1904

    PubMed  PubMed Central  Google Scholar 

  39. Martin-Ortigosa S, Wang K (2014) Proteolistics: a biolistic method for intracellular delivery of proteins. Transgenic Res 23:743–756

    CAS  PubMed  Google Scholar 

  40. 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 7:40

    PubMed  PubMed Central  Google Scholar 

  41. Nagata T (1987) Interaction of plant protoplast and liposome. In: Green R, Widder KJ (eds) Methods in Enzymology. Academic Press, Orlando, pp 34–39

    Google Scholar 

  42. Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. Int Rev Cytol 132:1–30

    CAS  Google Scholar 

  43. Paix A, Folkmann A, Rasoloson D, Seydoux G (2015) High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics 201:47–54

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Puchta H, Fauser F (2014) Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J 78:727–741

    CAS  PubMed  Google Scholar 

  45. Rademacher T, Sack M, Blessing D, Fischer R, Holland T, Buyel J (2019) Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering. Plant Biotechnol J. https://doi.org/10.1111/pbi.13081

    Article  PubMed  PubMed Central  Google Scholar 

  46. Ramakrishna S, Dad A-BK, Beloor J, Gopalappa R, Lee S-K, Kim H (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–1027

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Rees HA, Komor AC, Yeh W-H, Caetano-Lopes J, Warman M, Edge ASB, Liu DR (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8:15790

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Roest S, Gilissen LJW (1989) Plant regeneration from protoplasts: a literature review. Acta Bot Neerl 38:1–23

    Google Scholar 

  49. Roest S, Gilissen LJW (1993) Regeneration from protoplasts a supplementary literature review. Acta Bot Neerl 42:1–23

    Google Scholar 

  50. Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE, Haliburton GE, Ye CJ, Bluestone JA, Doudna JA, Marson A (2015) Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci USA 112:10437–10442

    CAS  PubMed  Google Scholar 

  51. Seki A, Rutz S (2018) Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J Exp Med 215:985–997

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Shaul O, Mironov V, Burssens S, Van Montagu M, Inze D (1996) Two Arabidopsis cyclin promoters mediate distinctive transcriptional oscillation in synchronized tobacco BY-2 cells. Proc Natl Acad Sci USA 93:4868–4872

    CAS  PubMed  Google Scholar 

  53. Staahl BT, Benekareddy M, Coulon-Bainier C, Banfal AA, Floor SN, Sabo JK, Urnes C, Munares GA, Ghosh A, Doudna JA (2017) Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol 35:431–434

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Stewart CN Jr, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14:748–750

    CAS  PubMed  Google Scholar 

  55. Subburaj S, Chung SJ, Lee C, Ryu S-M, Kim DH, Kim J-S, Bae S, Lee G-J (2016) Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35:1535–1544

    CAS  PubMed  Google Scholar 

  56. Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM (2016) Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun 7:13274

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Woo JW, Kim J, Kwon SII, Corvalan C, Cho SW, Kim H, Kim S-G, Kim S-T, Choe S, Kim J-S (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1165

    CAS  PubMed  Google Scholar 

  58. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:565–1572

    Google Scholar 

  59. Yu X, Liang X, Xie H, Kumar S, Ravinder N, Potter J, de Mollerat du Jeu X, Chesnut JD (2016) Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX. Biotechnol Lett 38:919–929

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu J-L, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank the Advance Research Projects Agency Energy (Award no. DE-AR0000331), Thermo Fisher. and the BioEnergy Science Center (BESC) for funding. The BESC was a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. We are also grateful for the funding from University of Tennessee and the Ivan Racheff endowment, and from North Carolina State University for the startup funds to the Liu laboratory. Research was enabled by Hatch grants. We thank Kacie Reynolds for her assistance with the miniprep work.

Author information

Affiliations

Authors

Contributions

WL, JDC, JPY, and CNS conceived and designed the project. WL, MM, MHC, RJM, CAO, and GAM conducted the experiments, and collected and analyzed the data. KPB developed the transgenic BY2 cells. WL, MM, JDC, and CNS wrote the manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Wusheng Liu or Charles Neal Stewart Jr..

Ethics declarations

Conflict of interest

The authors declare competing interests. The lipofection method is the subject of an invention disclosure and patent application at UTK. The University of Tennessee received funding from Thermo Fisher for the research and some of the authors are employees of the company.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Baochun Li.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 906 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, W., Rudis, M.R., Cheplick, M.H. et al. Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Rep 39, 245–257 (2020). https://doi.org/10.1007/s00299-019-02488-w

Download citation

Keywords

  • Protein delivery
  • Cas9/gRNA ribonucleoprotein
  • Lipofection
  • Biolistics
  • Genome editing
  • Protoplast
  • Tobacco
  • BY2