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

Citation

Gao, R., Feyissa, B.A., Croft, M., Hannoufa, A. (2018). Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa, 247(4), 1043-1050. http://dx.doi.org/10.1007/s00425-018-2866-1

Plain language summary

The CRISPR-Cas9 technology enables a variety of applications in plants as it can create point mutations within genes. Successful application and validation of the CRISPR technique in Medicago sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. In this study, we used CRISPR/Cas9 technique to mutate the SPL9 gene in alfalfa. Plants with relatively high genome editing rates were subjected to analysis by restriction enzyme digestion/PCR amplification analyses. 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.

Abstract

© 2018, Crown. 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.

Publication date

2018-04-01