Repositorium
Journal Article / 2018
Gao, Ruimin; Feyissa, Biruk A.; Croft, Mana; Hannoufa, Abdelali
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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
697 |
Hannoufa, Abdelali Canada |
Medicago sativa |
CRISPR/Cas9 SPL9 |
No information SDN1 |
Basic research Basic research |