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Planta

, Volume 247, Issue 4, pp 1043–1050 | Cite as

Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa

  • Ruimin Gao
  • Biruk A. Feyissa
  • Mana Croft
  • Abdelali Hannoufa
Short Communication

Abstract

Main conclusion

The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa).

RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because of the complex features of the alfalfa genome, we first used droplet digital PCR (ddPCR) for high-throughput screening of large populations of CRISPR-modified plants. Based on the results of genome editing rates obtained from the ddPCR screening, plants with relatively high rates were subjected to further analysis by restriction enzyme digestion/PCR amplification analyses. PCR products encompassing the respective small guided RNA target locus were then sub-cloned and sequenced to verify genome editing. In summary, we successfully applied the CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some insights into opportunities to apply this technology in future alfalfa breeding. The overall efficiency in the polyploid alfalfa genome was lower compared to other less-complex plant genomes. Further refinement of the CRISPR technology system will thus be required for more efficient genome editing in this plant.

Keywords

Alfalfa CRISPR/Cas9 Droplet digital PCR (ddPCR) Gene editing Multiplex mutagenesis 

Abbreviations

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

ddPCR

Droplet digital PCR

NHEJ

Non-homologous end joining

PAM

Protospacer-associated motif

sgRNA

Small guided RNA

SPL

Squamosa promoter binding protein like

Notes

Acknowledgements

This project was funded by a Grant (J-000260) from Agriculture and Agri-Food Canada to AH. RG was the recipient of a NSERC Visiting Fellowship to a Canadian Government Laboratory.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

425_2018_2866_MOESM1_ESM.docx (412 kb)
Supplementary material 1 (DOCX 412 kb)
425_2018_2866_MOESM2_ESM.docx (75 kb)
Supplementary material 2 (DOCX 74 kb)
425_2018_2866_MOESM3_ESM.xlsx (11 kb)
Supplementary material 3 (XLSX 11 kb)

References

  1. Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573.  https://doi.org/10.1038/nature13579 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Arshad M, Feyissa BA, Amyot L, Aung B, Hannoufa A (2017) MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13. Plant Sci 258:122–136.  https://doi.org/10.1016/j.plantsci.2017.01.018 CrossRefPubMedGoogle Scholar
  3. Aung B, Gruber MY, Amyot L, Omari K, Bertrand A, Hannoufa A (2015) MicroRNA156 as a promising tool for alfalfa improvement. Plant Biotechnol J 13:779–790.  https://doi.org/10.1111/pbi.12308 CrossRefPubMedGoogle Scholar
  4. Badhan A, Jin L, Wang Y et al (2014) Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility. Biotechnol Biofuels 7:39.  https://doi.org/10.1186/1754-6834-7-39 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712.  https://doi.org/10.1126/science.1138140 CrossRefPubMedGoogle Scholar
  6. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551–2561.  https://doi.org/10.1099/mic.0.28048-0 CrossRefPubMedGoogle Scholar
  7. Braatz J, Harloff HJ, Mascher M, Stein N, Himmelbach A, Jung C (2017) CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol 174:935–942.  https://doi.org/10.1104/pp.17.00426 CrossRefPubMedGoogle Scholar
  8. Brocal I, White RJ, Dooley CM et al (2016) Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish. BMC Genom 17:259.  https://doi.org/10.1186/s12864-016-2563-z CrossRefGoogle Scholar
  9. Cai Y, Chen L, Liu X et al (2015) CRISPR/Cas9-mediated genome editing in soybean hairy roots. PLoS One 10:e0136064.  https://doi.org/10.1371/journal.pone.0136064 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Doench JG, Fusi N, Sullender M et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191.  https://doi.org/10.1038/nbt.3437 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dominguez AA, Lim WA, Qi LS (2016) Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17:5–15.  https://doi.org/10.1038/nrm.2015.2 CrossRefPubMedGoogle Scholar
  12. Fauser F, Roth N, Pacher M, Ilg G, Sanchez-Fernandez R, Biesgen C, Puchta H (2012) In planta gene targeting. Proc Natl Acad Sci USA 109:7535–7540.  https://doi.org/10.1073/pnas.1202191109 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Feng Z, Mao Y, Xu N et al (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637.  https://doi.org/10.1073/pnas.1400822111 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Findlay SD, Vincent KM, Berman JR, Postovit LM (2016) A digital PCR-based method for efficient and highly specific screening of genome edited cells. PLoS One 11:e0153901.  https://doi.org/10.1371/journal.pone.0153901 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gao R, Austin RS, Amyot L, Hannoufa A (2016) Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa. BMC Genom 17:658.  https://doi.org/10.1186/s12864-016-3014-6 CrossRefGoogle Scholar
  17. Guell M, Yang L, Church GM (2014) Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA). Bioinformatics 30:2968–2970.  https://doi.org/10.1093/bioinformatics/btu427 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649:247–256.  https://doi.org/10.1007/978-1-60761-753-2_15 CrossRefPubMedGoogle Scholar
  19. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:e188.  https://doi.org/10.1093/nar/gkt780 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Krishnakumar V, Kim M, Rosen BD, Karamycheva S, Bidwell SL, Tang H, Town CD (2015) MTGD: the Medicago truncatula genome database. Plant Cell Physiol 56:e1.  https://doi.org/10.1093/pcp/pcu179 CrossRefPubMedGoogle Scholar
  21. Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7:1494–1496.  https://doi.org/10.1093/mp/ssu044 CrossRefPubMedGoogle Scholar
  22. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Liang Z, Zhang K, Chen K, Gao C (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
  24. Marraffini LA, Sontheimer EJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463:568–571.  https://doi.org/10.1038/nature08703 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Meng Y, Hou Y, Wang H et al (2016) Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula. Plant Cell Rep.  https://doi.org/10.1007/s00299-016-2069-9 PubMedGoogle Scholar
  26. Michno JM, Wang X, Liu J, Curtin SJ, Kono TJ, Stupar RM (2015) CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food 6:243–252.  https://doi.org/10.1080/21645698.2015.1106063 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Miyaoka Y, Chan AH, Judge LM et al (2014) Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11:291–293.  https://doi.org/10.1038/nmeth.2840 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mock U, Machowicz R, Hauber I et al (2015) mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic Acids Res 43:5560–5571.  https://doi.org/10.1093/nar/gkv469 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Mock U, Hauber I, Fehse B (2016) Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nat Protoc 11:598–615.  https://doi.org/10.1038/nprot.2016.027 CrossRefPubMedGoogle Scholar
  30. 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 31:691–693.  https://doi.org/10.1038/nbt.2655 CrossRefPubMedGoogle Scholar
  31. Perez EE, Wang J, Miller JC et al (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26:808–816.  https://doi.org/10.1038/nbt1410 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sanderson MA, Adler PR (2008) Perennial forages as second generation bioenergy crops. Int J Mol Sci 9:768–788.  https://doi.org/10.3390/ijms9050768 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Schmid-Burgk JL, Schmidt T, Gaidt MM, Pelka K, Latz E, Ebert TS, Hornung V (2014) OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines. Genome Res 24:1719–1723.  https://doi.org/10.1101/gr.176701.114 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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 CrossRefPubMedGoogle Scholar
  35. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67.  https://doi.org/10.1038/nature13011 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Thomas HR, Percival SM, Yoder BK, Parant JM (2014) High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis. PLoS One 9:e114632.  https://doi.org/10.1371/journal.pone.0114632 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Tian L, Wang H, Wu K, Latoszek-Green M, Hu M, Miki B, Brown DCW (2002) Efficient recovery of transgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker gene expression. Plant Cell Rep 20:1181–1187CrossRefGoogle Scholar
  38. Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in Genome Editing and Beyond. Ann Rev Biochem 85(1):227–264CrossRefPubMedGoogle Scholar
  39. Upadhyay SK, Kumar J, Alok A, Tuli R (2013) RNA-guided genome editing for target gene mutations in wheat. G3 (Bethesda) 3:2233–2238.  https://doi.org/10.1534/g3.113.008847 CrossRefGoogle Scholar
  40. Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6:1975–1983.  https://doi.org/10.1093/mp/sst119 CrossRefPubMedGoogle Scholar
  41. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Xu R, Li H, Qin R, Wang L, Li L, Wei P, Yang J (2014) Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice (N Y) 7:5.  https://doi.org/10.1186/s12284-014-0005-6 CrossRefGoogle Scholar

Copyright information

© Crown 2018

Authors and Affiliations

  1. 1.Agriculture and Agri-Food CanadaLondonCanada
  2. 2.Department of BiologyUniversity of Western OntarioLondonCanada

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