Master of Science
CRISPR is a well-known adaptive defense mechanism that gained attention through its ability to be easily reprogrammable. Over time, the native CRISPR-Cas system evolved to tolerate mismatches, broadening its cleavage preferences; however, this development poses concerns with off-target cleavage in gene editing. Here, we introduced a D10E mutation into SaCas9 to potentially reduce off-target cleavage. To characterize SaCas9[D10E] and its tolerance for mutations, we designed 21 different substrates that each contained a single transversion in a therapeutically relevant EMX1-1gene. Through a series of in vitro cleavage assays, SaCas9[WT] and SaCas9[D10E] were compared across these substrates. A kinetic analysis of SaCas9[D10E] revealed trends in initial cleavage rates as PAM proximal mutations exhibited reduced cleavage activity and PAM distal mutations displayed enhanced activity. Furthermore, the ability of SaCas9[D10E] was highlighted through a competition assay that displayed discrimination between single nucleotide differences.
Summary for Lay Audience
CRISPR is an adaptive defense mechanism that originated in bacteria and is currently utilized as a gene editing tool. In bacteria, when a virus invades the cell, CRISPR functions via guided targeted protein-RNA complex that generates a double stranded break in inserted viral DNA to destroy it. Since then, CRISPR has evolved to tolerate mutations to broaden its cleavage specificity. While broadening its cleavage specificity is good for bacteria, it results in off-target cleavage, which reduces its gene editing capabilities. The focus of my thesis is to find a solution to its tolerance and improve its accuracy in gene editing.
There are many pathways that we can take to reduce off-target cleavage, but, here, we chose to generate a conservative mutation of D10E in Cas9 to alter cleavage specificity and hypothesized that it can potentially reduce off-target cleavage.
This thesis established that the SaCas9[D10E] variant is unable to reduce off-target cleavage. Rather, D10E can discriminate between single mutations of on and off targets while also highlighting trends in cleavage rates of SaCas9[D10E].
Zhang, Claire, "Kinetic Analyses of SaCas9[D10E] in vitro" (2023). Electronic Thesis and Dissertation Repository. 9816.
Available for download on Sunday, September 01, 2024