RNA-guided genome engineering using CRIPR-Cas9 enables a variety of
applications in plants. Successful application and validation of the CRISPR
technique in a multiplex genome, such as that of alfalfa will lead to major
advances in the improvement of this crop.

The team of Ruimin Gao from Agriculture and Agri-Food Canada used the
CRISPR-Cas9 system to target the squamosa promoter binding protein like 9
(SPL9) gene in alfalfa. Due to the complexity of the alfalfa genome, the
team first screened large populations of CRISPR-modified plants to select
plants with high genome editing rates. These plants were then subjected to
further analyses and mutations on the desired target site were verified in
these selected plants.

The team successfully applied the CRISPR-Cas9 technique to edit the SPL9
gene in a multiplex alfalfa genome. However, the overall efficiency of
CRISPR in the polyploid alfalfa genome was lower compared to other plant
genomes. Further refinement of the CRISPR technology system will be required
for more efficient genome editing in alfalfa.



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