, 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 HannoufaEmail author
Short Communication


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.


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



Clustered Regularly Interspaced Short Palindromic Repeats


Droplet digital PCR


Non-homologous end joining


Protospacer-associated motif


Small guided RNA


Squamosa promoter binding protein like



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

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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)


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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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