Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa
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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.
KeywordsAlfalfa CRISPR/Cas9 Droplet digital PCR (ddPCR) Gene editing Multiplex mutagenesis
Clustered Regularly Interspaced Short Palindromic Repeats
Droplet digital PCR
Non-homologous end joining
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.
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