Overview and Motivation

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 disease 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. We express two guide RNAs that target adjacent regions within 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.

Testing the Sensor:
We fused C or N terminal components of the split venus protein to the C-terminal of the CAS9. We designed guide RNAs that target a defective eGFP sequence. Cells were transfected with both fusion proteins, the two unique guide RNAs 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 guide RNA were transfected.

Goals:
A DNA sensor that is easily customizable 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.

Cas9 Fusion Background Recombination

We transfected constitutively expressed CAS9-venus fusions without a guide RNA and looked at levels of background fluorescence in order to understand the effect fusing CAS9 to the venus proteins had on the ability of venus to reconstitute. We confirmed that our venus proteins we still functional when fused to the CAS9 and gained an understanding of the amount of DNA we needed to transfect to start seeing fluorescence.

DNA Sensing

We transfected the Cas9-Venus fusions, target plasmid, and different guide RNA combinations. We used flow cytometry analysis to look for an increase in yellow fluorescence due to the reconstitution of the venus proteins. Experimental wells were compared to the background expression of Cas9-Venus that is not specifically targeted to the plasmid. The Cas9 fusions bind on opposing DNA strands. The best results of DNA sensing were seen when the guide RNA sequences were 25-60bp apart. Future optimization is required to determine the best configuration of guideRNA sequences to maximize probability of split venus reconstitution.
The Circuit: