Electronic Thesis and Dissertation Repository

Thesis Format



Master of Science




Edgell, David R.


CRISPR-Cas9 nucleases are commonly used for genome editing but are limited by their off- target effects, which must be reduced for therapeutic use. Current strategies for engineering higher fidelity variants do not directly compare on- versus off-target activity. I establish Directed Evolution Using Fluorescence Activated Cell Sorting (DEUFACS), a bacterial dual- fluorophore system for characterizing and altering the sequence preferences of nucleases. I aimed to use DEUFACS to identify SaCas9 variants with increased on-target and decreased off-target activity. I found that D10E substitution in the RuvC domain of SaCas9 improved discrimination and was superior to other RuvC active site variants containing substitutions with similar properties. Using tiled transversion substrates, SaCas9[D10E] preferentially cleaved the on-target versus most transversions. However, D10E did not show enriched on-target activity using a D10 site-saturation library. My results suggest DEUFACS is a powerful tool for the simultaneous profiling of nuclease activity on desired on-target versus unwanted off- target substrates.

Summary for Lay Audience

Genome editing involves manipulating DNA sequences to change the instructions in our DNA. The field has tremendous potential in agriculture, biotechnology and medicine for the treatment of genetic diseases. The main tool for genome-editing is CRISPR-Cas9, a kind of genetic scissor. It relies on an RNA component that guides it to the right spot in our DNA and a Cas9 enzyme and two blades to make the cut in DNA. Our cells can then fix this cut, which lets us correct, replace, or turn off genes. However, problems arise because sometimes the genetic scissors can cut in the wrong places, which poses safety concerns. This is particularly important in fields like medicine where minimizing health risks to undetectable levels is of utmost importance. Scientists are trying to make these scissors more precise but are limited in available methods for making new variants that directly compare how many cuts are right versus wrong.

In this thesis, I develop a bacterial system relying on fluorescence, Directed Evolution Using Fluorescence Activated Cell Sorting (DEUFACS), that helps us identify if the scissors are cutting where they should be and at the same time identifies if they cut in places they should not. I used this system to characterize a new version of the scissors with a modification, E instead of D at position 10 (D10E), in the region responsible for making the cut, which is essentially in one of the scissor’s blades. I hypothesized that this modification would make the scissors more precise as it had this effect on other types of scissors with a similar mechanism of cutting. Using the new DEUFACS system, I found that the D10E modification was better at distinguishing between right and wrong cuts compared to the original scissors and similar modifications introduced in different regions of the same blade. However, the D10E scissors cut less overall. I also looked at how single changes in the DNA around the cut to simulate wrong sequences affected the D10E scissor’s precision compared to original scissors. With most of these changes in DNA, D10E preferred to cut the right sequence, while the original scissors cut both the right and wrong sequence. Interestingly, changes towards the end of the blade furthest from the handle resulted in D10E changing its preference for the wrong sequence. Lastly, I tried introducing all possible changes in the same position of the scissors and tested them at the same time to see if substituting E instead of D was the best choice. The D to E change did not work as well in this mix of all possible changes as it did not cut much.

These results establish the new DEUFACS system as a powerful tool for characterizing the specificity of genetic scissors and has the potential to be used to make them even better. The importance of directly comparing right and wrong cuts is highlighted as we need to ensure not to make the scissors too weak while trying to make them safer.

Available for download on Monday, September 30, 2024

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Biochemistry Commons