Gibson Assembly Attempts

After designing the set of sgRNA sequences to target different highly conserved spots on the CAMV viral genome, we found that it would not be possible to synthesize them all as one large fragment because of the sequence-repeat limitations of the synthesis procedure at the sequencing company IDT. It was decided that we'd order the individual sgRNAs one by one, piece them together along with the Cas9 coding sequence, and insert that entire fragment into our Agrobacterium-Arabidopsis transfer vector (pCAMBIA) using Gibson Assembly. Since we were attempting an ambitious assembly project (six fragments, two of which were >5kb), none of the attempts worked. Thankfully, the homologous ends that each fragment was designed to have lent themselves to other forms of cloning which were not necessarily Gibson-based. PCR overlap extension -- a technique suggested by the University of Ottawa iGEM team -- provided us with the route to success. By joining together some of the smaller fragments into clusters, we sought to increase the probably of Gibson assembly working. Two fragments of the five-fragment Gibson Assembly plan were successfully joined by PCR overlap extension before another Gibson reaction was attempted. The now four-fragment reaction produced colonies that have produced promising PCR diagnostic results. Since plants take time to grow, after the integration of our construct into the Arabidopsis genome by Agrobacterium-mediated floral dip we'll need to wait approximately 1-2 months for mature CRISPR-Arabidopsis plants to be in a testable state against the virus.