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 PAM 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 but instead 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) with both an NGG PAM sequence and an NGAG PAM sequence.

Experimental Design

All of our experiments to study GFP 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. By measuring an average fluorescence intensity of cells, we can determine the average intensity of a cell population.

Our first step was to create the EQR variant in dCas9 by using site directed mutagenesis (reference). This was done on a dCas9 plasmid ordered from AddGene. Two other plasmids were constructed with both a gene producing GFP and an sgRNA target recognizing either an NGG or an NGAG PAM site on the GFP promoter J23101 (link?).

Constructs

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

Results