Advertisement

Deep sequencing analysis of CRISPR/Cas9 induced mutations by two delivery methods in target model genes and the CENH3 region of red cabbage (Brassica oleracea var. capitata f. rubra)

  • Ester Stajič
  • Agnieszka Kiełkowska
  • Jana Murovec
  • Borut BohanecEmail author
Original Article
  • 61 Downloads

Abstract

CRISPR/Cas9 is a versatile and highly efficient genome editing tool used in many different plant species. In the present study, we compared the two most commonly used transient expression methods for genome editing, protoplast transfection and infiltration of Agrobacterium tumefaciens, to develop a rapid and efficient validation protocol. Vectors designed to target four different sites in the cabbage genome (two of which were model target genes and two related to the centromere-specific histone H3 (CENH3) gene) were delivered to two red cabbage cultivars, ‘Huzaro F1’ and ‘Rebecca F1’. Targeted deep sequencing analysis showed that CRISPR/Cas9 vectors induced mutations in both cultivars at all target sites and revealed mutation rates of 1.27–11.95% for protoplast transfection and 0.07–14.42% for agroinfiltration. Our results demonstrate successful genome editing in cabbages with CRISPR/Cas9 by two different approaches for the rapid evaluation of genome editing efficiency.

Key message

Comparison of two different transient transformation methods for the validation of sgRNA in red cabbage (B. oleracea var. capitata f. rubra).

Keywords

CRISPR/Cas9 Brassica oleracea Agroinfiltration Protoplast transfection Genome editing 

Notes

Acknowledgements

This work was supported by the Slovenian Research Agency (research programme P4-0077 and PhD student grant 1000-14-0510).

Author contributions

BB and JM conceived and designed the research, ES performed the experiments. ES and JM analysed the results, AK contributed to development of protoplast isolation protocol. ES wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Andrieu A, Breitler JC, Siré C et al (2012) An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves. Rice 5:1–13.  https://doi.org/10.1186/1939-8433-5-23 CrossRefGoogle Scholar
  2. Belhaj K, Chaparro-Garcia A, Kamoun S et al (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84.  https://doi.org/10.1016/j.copbio.2014.11.007 CrossRefGoogle Scholar
  3. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52.  https://doi.org/10.1016/j.biotechadv.2014.12.006 CrossRefGoogle Scholar
  4. Braatz J, Harloff HJ, Mascher M et al (2017) CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homologous gene copies in polyploid oilseed rape (Brassica napus L.). Plant Physiol 174:935–942.  https://doi.org/10.1104/pp.17.00426 CrossRefGoogle Scholar
  5. Brooks C, Nekrasov V, Lippman ZB et al (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 system. Plant Physiol 166:1292–1297.  https://doi.org/10.1104/pp.114.247577 CrossRefGoogle Scholar
  6. Che P, Anand A, Wu E et al (2018) Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application. Plant Biotechnol J 16:1388–1395.  https://doi.org/10.1111/pbi.12879 CrossRefGoogle Scholar
  7. Clement K, Rees H, Canver MC et al (2019) CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37(3):224–226.  https://doi.org/10.1038/s41587-019-0032-3 CrossRefGoogle Scholar
  8. Daud NFA, Hasbullah NA, Azis NA et al (2015) In vitro regeneration of Brassica oleracea var. capitata through stems, roots, leaves, and petioles cultures. International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7–8, 2015 Phuket. http://doi.org/10.15242/IICBE.C0415004
  9. Davey MR, Anthony P, Power JB et al (2005) Plant protoplast technology: current status. Acta Physiol Plant 27:117–129.  https://doi.org/10.1007/s11738-005-0044-0 CrossRefGoogle Scholar
  10. Doench JG, Hartenian E, Graham DB et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32:1261–1267.  https://doi.org/10.1038/nbt.3026 CrossRefGoogle Scholar
  11. Feng Z, Zhang B, Ding W et al (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23:1229–1232.  https://doi.org/10.1038/cr.2013.114 CrossRefGoogle Scholar
  12. Fu Y, Sander JD, Reyon D et al (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32:279–284.  https://doi.org/10.1038/nbt.2808 CrossRefGoogle Scholar
  13. Gao J, Wang G, Ma S et al (2015) CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol 87:99–110.  https://doi.org/10.1007/s11103-014-0263-0 CrossRefGoogle Scholar
  14. Glimelius K (1984) High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae. Physiol Plant 61:38–44.  https://doi.org/10.1111/j.1399-3054.1984.tb06097.x CrossRefGoogle Scholar
  15. Graf R, Li X, Chu VT et al (2019) sgRNA sequence motifs blocking efficient CRISPR/Cas9-mediated gene editing. Cell Rep 26:1098–1103.  https://doi.org/10.1016/j.celrep.2019.01.024 CrossRefGoogle Scholar
  16. Jia H, Wang N (2014) Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS ONE 9:e93806.  https://doi.org/10.1371/journal.pone.0093806 CrossRefGoogle Scholar
  17. Jiang W, Zhou H, Bi H et al (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:1–12.  https://doi.org/10.1093/nar/gkt780 CrossRefGoogle Scholar
  18. Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821.  https://doi.org/10.1126/science.1225829 CrossRefGoogle Scholar
  19. Kiełkowska A, Adamus A (2012) An alginate-layer technique for culture of Brassica oleracea L. protoplasts. Vitro Cell Dev Biol Plant 48:265–273.  https://doi.org/10.1007/s11627-012-9431-6 CrossRefGoogle Scholar
  20. Kirchner TW, Niehaus M, Debener T et al (2017) Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata. PLoS ONE 12:e0185429.  https://doi.org/10.1371/journal.pone.0185429 CrossRefGoogle Scholar
  21. Klimek M, Tomasz C, Levi O et al (2018) Efficient CRISPR/Cas9-based genome editing in carrot cells. Plant Cell Rep 37:575–586.  https://doi.org/10.1007/s00299-018-2252-2 CrossRefGoogle Scholar
  22. Lawrenson T, Shorinola O, Stacey N et al (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258.  https://doi.org/10.1186/s13059-015-0826-7 CrossRefGoogle Scholar
  23. Li JF, Norville JE, Aach J et al (2013) Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691.  https://doi.org/10.1038/nbt.2654 CrossRefGoogle Scholar
  24. Li C, Hao M, Wang W et al (2018) An efficient CRISPR/Cas9 platform for rapidly generating simultaneous mutagenesis of multiple gene homologs in allotetraploid oilseed rape. Front Plant Sci 9:442.  https://doi.org/10.3389/fpls.2018.00442 CrossRefGoogle Scholar
  25. Liang Z, Zhang K, Chen K et al (2014) Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J Genet Genom 41:63–68.  https://doi.org/10.1016/j.jgg.2013.12.001 CrossRefGoogle Scholar
  26. Lin CS, Hsu CT, Yang LH et al (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16:1295–1310.  https://doi.org/10.1111/pbi.12870 CrossRefGoogle Scholar
  27. Ma J, Xiang H, Meng DJDF (2017) Genome editing in potato plants by agrobacterium-mediated transient expression of transcription activator-like effector nucleases. Plant Biotechnol Rep 11:249–258.  https://doi.org/10.1007/s11816-017-0448-5 CrossRefGoogle Scholar
  28. Ma C, Zhu C, Zheng M et al (2019) CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system. Hortic Res 6:1–15.  https://doi.org/10.1038/s41438-018-0107-1 CrossRefGoogle Scholar
  29. Manavella PA, Chan RL (2009) Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies. Nat Protoc 4:1699–1707.  https://doi.org/10.1038/nprot.2009.178 CrossRefGoogle Scholar
  30. Menczel L, Nagy F, ZsR Kiss et al (1981) Streptomycin resistant and sensitive hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195.  https://doi.org/10.1007/BF00264975 CrossRefGoogle Scholar
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497.  https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
  32. Murovec J, Guček K, Bohanec B et al (2018) DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes. Front Plant Sci 9:1–9.  https://doi.org/10.3389/fpls.2018.01594 CrossRefGoogle Scholar
  33. Nekrasov V, Staskawicz B, Weige D et al (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693.  https://doi.org/10.1038/nbt.2655 CrossRefGoogle Scholar
  34. Park J, Bae S, 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 CrossRefGoogle Scholar
  35. Park J, Lim K, Kim JS et al (2017) Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33:286–288.  https://doi.org/10.1093/bioinformatics/btw561 CrossRefGoogle Scholar
  36. Ravi M, Chan SW (2010) Haploid plants produced by centromere-mediated genome elimination. Nature 464:615–618.  https://doi.org/10.1038/nature08842 CrossRefGoogle Scholar
  37. Sahab S, Hayden MJ, Mason J et al (2019) Mesophyll protoplasts and PEG-mediated transfections transient assays and generation of stable transgenic canola plants. In: Kumar S, Barone P, Smith M (eds) Transgenic plants. Methods in molecular biology, vol 1864. Humana Press, New York.  https://doi.org/10.1007/978-1-4939-8778-8_10 Google Scholar
  38. Sander JD, Joung JK (2014) CRISPR-Cas system for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355.  https://doi.org/10.1038/nbt.28422 CrossRefGoogle Scholar
  39. Shan Q, Wang Y, Li J et al (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688.  https://doi.org/10.1038/nbt.2650 CrossRefGoogle Scholar
  40. Shan Q, Wang Y, Li J et al (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9:2395–2410.  https://doi.org/10.1038/nprot.2014.157 CrossRefGoogle Scholar
  41. Subburaj S, Chung SJ, Lee C et al (2016) Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35:1535–1544.  https://doi.org/10.1007/s00299-016-1937-7 CrossRefGoogle Scholar
  42. Wang T, Wei JJ, Sabatini DM et al (2014a) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84.  https://doi.org/10.1126/science.1246981 CrossRefGoogle Scholar
  43. Wang Y, Cheng X, Shan Q et al (2014b) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951.  https://doi.org/10.1038/nbt.2969 CrossRefGoogle Scholar
  44. Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato, and Arabidopsis. Plant Biotechnol J 3:259–273.  https://doi.org/10.1111/j.1467-7652.2005.00123.x CrossRefGoogle Scholar
  45. Xing HL, Dong L, Wang ZP et al (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327.  https://doi.org/10.1186/s12870-014-0327-y CrossRefGoogle Scholar
  46. Yang H, Wu JJ, Tang T et al (2017) CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci Rep 7:7489.  https://doi.org/10.1038/s41598-017-07871-9 CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Ester Stajič
    • 1
  • Agnieszka Kiełkowska
    • 2
  • Jana Murovec
    • 1
  • Borut Bohanec
    • 1
    Email author
  1. 1.Department of Agronomy, Biotechnical FacultyUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Department of Genetics, Plant Breeding and Seed ScienceUniversity of Agriculture in KrakowKrakowPoland

Personalised recommendations