Team:Waterloo/Results
Results
Project Results
One of the major results that Waterloo iGEM had accomplished over the summer 2015 is showing that adding restriction sites and a single mutation in the scaffold region of the sgRNA did not affect the functionality of the sgRNA in CRISPR dCas9 system. The assay used for this experiment is amnis Imaging Flow Cytometry.
In addition, we accomplished to mutate dCas9 at three sites 1135, 1335, and 1337 to alter its PAM site from native NGG to NGAG using the Quick Change protocol. The mutations were confirmed using sequencing. Preliminary experiments were done using sgRNA targeting the J23101 promoter upstream of the GFP using NGAG PAM site. Further result analysis is needed to confirm the functionality of the mutated dCas9.
We were finally able to show that Cas9 was expressed from protoplasts using anti-Cas9 antibodies and dot blots. Additionally, we were able to agroinfiltrate our tentative pCAMBIA + Cas9 + sgRNA vector into Arabidopsis .