By: Dr. Rakhi Rajan - University of Oklahoma
CRISPR-Cas systems protect bacteria and archaea from foreign genetic elements based on sequence specific RNA-guided targeting and cleavage of the intruder genome by signature Cas nucleases. Cas9 and Cas12a are two CRISPR endonucleases, comprised of multi-domain enzymes that exhibit intricate conformational controls to elicit sequence-specific DNA cleavage. Even though Cas nucleases have revolutionized biotechnological and gene therapy applications, the methodology suffers from the ability of Cas nucleases to cleave targets that are not completely complementary to the guide-region of the RNA. To address these off-target effects, we have focused on identifying the role of a highly-conserved arginine-rich helix present in several Cas nucleases, called the bridge helix, in regulating specificity of DNA cleavage. Our results identified a novel allosteric control that the bridge helix exerts on the endonucleases sites of Cas9, which is directed by a loop-to-helical transition in the bridge helix. The mechanism is conserved in Cas12a as well, establishing that bridge helix variations can be used to develop high-fidelity Cas protein variants for genome applications.
Another aspect of our research is to address promiscuous DNA cleavages by Cas nucleases. We discovered that several Cas nucleases possess non-specific DNA cleavage in the absence of a guide-RNA. Our on-going research shows that manipulating the active site of Cas nucleases, specifically related to divalent metal ion binding, can remove promiscuous DNA cleavage without significantly reducing RNA guided DNA cleavage. We propose that removing off-target and promiscuous DNA cleavages are crucial in deploying the full potential of Cas nucleases in gene therapy and other genome applications. Our combinatorial approach including protein engineering, biochemistry, kinetic modeling, and molecular dynamics simulations provide translatable approaches to study Cas nucleases.
The talk will also include information about graduate studies in the Department of Chemistry and Biochemistry at the University of Oklahoma.