Team:Waterloo/Lab/dCas9
dCas9 Modification
One of our goals this year was to engineer a version of the Cas9 protein that could recognize different protospacer adjacent motifs (PAMs) other than the regular NGG. Kleinstiver et al (reference) recently published data on Cas9 mutants that effectively cut unique PAM sites. A specific EQR variant in the publication recognized a NGAG site with three amino acids mutated. Our goal was to try and replicate these mutations with dCas9. dCas9 is commonly used since it represses a gene rather than cutting. Target sites were designed in promoters of green fluorescent protein (GFP) and red fluorescent protein (RFP) with both a NGG PAM sequence and a NGAG PAM sequence.