(491.3) Examining the Interaction Between Cas9 and Ku, and its Implications for Gene Editing

Poster Board Number: A111

Catherine Hayes (University of Illinois at Chicago), Aditya Bharti (University of Illinois at Chicago), Les Hanakahi (University of Illinois at Chicago, University of Illinois at Chicago)

Gene editing via CRISPR/Cas9 is an innovative new tool that allows scientists and clinicians to alter DNA. This can be done by inserting new DNA or by knocking out the function of preexisting DNA. Both processes begin with Cas9, an enzyme that acts as molecular scissors to introduce a DNA double-strand break (DSB) at a specific location. The way this DSB is repaired determines whether new DNA is inserted or preexisting DNA is knocked out.

Insertion of the new DNA sequence relies on a DNA-repair process called homologous recombination. Knocking out the function of a gene is achieved through the DNA-repair process non-homologous end joining (NHEJ). Unfortunately, the majority of Cas9-induced DSBs are repaired by NHEJ, thereby posing a barrier to inserting new genes. So why is NHEJ so prominent at Cas9-induced DSBs?

We have evidence that Cas9 interacts with the NHEJ initiating factor Ku. We, and others, used lysates from Cas9-expressing human cells and found that Cas9 co-immunoprecipitated with Ku. These observations may reflect either a direct Ku-Cas9 protein-protein interaction, or an indirect interaction mediated by other human cellular proteins, DNA, or both.

I hypothesize that Cas9 and Ku have a direct physical interaction. To test my hypothesis, I carried out affinity-capture assays using purified recombinant proteins. I found that Cas9 captured Ku, indicating that the interaction is not dependent on other human cellular proteins. This interaction also occurred when Ku-DNA interactions are disrupted by adding ethidium bromide or benzonase. This indicates that the interaction is not due to the DNA-binding capabilities of Ku.

These experiments indicate that Ku and Cas9 have a direct protein-protein interaction. Because Ku initiates NHEJ, our results suggest the possibility that the Cas9-Ku interaction may recruit NHEJ factors to Cas9-induced DSBs and provides a plausible explanation of the bias towards NHEJ repair of Cas9-induced DSBs.

Support or Funding Information

This research was funded through the Riback Fellowship from the University of Illinois at Chicago College of Pharmacy and through the Chicago Biomedical Consortium. Special thanks to Stephen J. Kron, MD PhD from the University of Chicago for insight and to Cayla Kleeman for technical assistance and instruction.



Schematic representation of possible mechanisms for observed interaction between Ku and Cas9 during co-immunoprecipitation assays. I hypothesize that a direct interaction is the mechanism of this interaction.