CRISPy Lettuce
Engineering
BME 129
The CRISPy Lettuce group genetically modified Salinas Lettuce using CRISPR/Cas9 to immunize it from Lettuce Mosaic Virus (LMV) through a knockout of the eIF4E gene. This potyvirus is not harmful to people, but it makes lettuce unmarketable and causes loss to the entire crop yield due to LVM’s ability to easily spread through aphids and seeds from infected plants. LVM requires stabilization from eukaryotic initiation factor (eIF4E) to replicate, so the gene of this protein was the target for modification. A CRISPR/Cas9 plasmid with our own designed sgRNA targeting eIF4E and Cas9 sequence was cloned into Agrobacterium tumefaciens. Cas9 was assembled in the plant cell and used the sgRNA to cleave the gene, inducing DNA repair through non-homologous end joining. Once the Agrobacterium took up the extracellular DNA, they were dripped onto the lettuce cotyledons in order to transfer the plasmid from bacteria to plant. The sgRNA and the Cas9 structure from the plasmid then recognized a portion of the eIF4E gene and inactivated it so that LMV cannot replicate within the plant cell thus giving the lettuce immunity.
