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 after three different amino acids in the original Cas9 were changed. The EQR variant includes the changes D1135E, R1335Q, and T1337R. Our goal was to try and replicate these mutations with dCas9, which is commonly used since it represses a gene rather than cutting one. 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.

Experimental Design

All of our experiments to study GFP and RFP expression with and without addition of dCas9 were performed on the Amnis Image Stream machine (Amnis 2015). The Image Stream is a fluorescence microscope combined with a flow cytometer, letting one to perform a quantitative method of fluorescence detection while visualizing the actual cells.

Constructs

326 GFP NGAG, Target 1.0 with GFP control, Mutated dCas9, Original dCas9 from Addgene (reference)

Results