Overview and Motivation

We hope to use exosome-mediated cell-to-cell communication to actuate a signal in naïve cells. The ability to detect the presence of specific DNA sequences in a naïve cell could be very useful. Therefore we design a novel DNA sensor that utilizes the Cas9/CRISPR system that can hopefully be packaged into an exosome and transported to a naïve cell. DNA sensors can be used to detect the presence of pathogenic and genetic diseases. The sensor binds to a specific DNA sequence and generates a detectable output when the target is found. Creating a standard DNA sensor that can be used to detect the presence of a variety of diseases caused by foreign DNA or modified DNA is a useful tool for disease diagnosis.

Enabling Technology:
We propose a novel DNA sensor that utilizes the DNA binding capabilities of the CAS9 protein. The CAS9/ CRISPR system is easily modified to target any DNA sequence of interest. We fused CAS9 to split Venus florescence proteins. The Venus fluorescence acts as the detectable output of DNA targeting. To guide Cas9 to the target plasmid we expressed two guide RNAs (gRNA) under a U6 promoter. The gRNAs are complementary to adjacent segments of the target sequence. Targeting CAS9-split Venus fusions to the same region should increase the probability of fluorescent protein reconstitution. Therefor if our target sequence is presence, we will see yellow fluorescence.

Goals:
A DNA sensor that is easily customized to detect a variety of diseases would be an especially powerful tool if we could package the components of the sensor and transport them to a naive cell. Our ultimate goal would be to target CAS9 and our DNA sensing system to exosomes.

Leucine Zipper Fusion

To confirm that increasing the proximity of split venus parts increased the probability of protein reconstitution, we constitutively expressed split venus leucine zipper fusions. Leucine zippers are composed of c-fos and c-jun domains. These proteins usually dimerize. Split venus proteins with zippers are pulled closer together due to interactions between the c-jun and c-fos domains thus increasing the probability of protein reconstitution. Our transfection also showed us that the background fluorescence of split venus proteins without leucine zippers is fairly low relative to the increase in fluoresce we see with the addition of zippers. We hope to see a similar increase in yellow fluorescence when Cas9 is used to increase the proximity between split venus components.

DNA Sensing

We fused C or N terminus components of the split venus protein to the C-terminus of CAS9. We designed gRNAs that target a defective eGFP sequence. Cells were transfected with both fusion proteins, the two unique gRNAs that guide the CAS9 to adjacent DNA sequences, and the target plasmid. Flow cytometry data was collected and analyzed to determine if there was an increase in yellow fluorescence when compared to a control in which CAS9-venus fusions with no gRNA were transfected.

The Cas9 fusions bind on opposing DNA strands. The best results of DNA sensing were seen when the gRNA sequences were 25-60bp apart. Future optimization is required to determine the best configuration of gRNA sequences to maximize probability of split venus reconstitution.