Generation of Conditional Ku70 Knockouts in Human Cell Lines Using CRISPR/Cas9 and Dual-Nuclease CRISPR/TevCas9
Master of Science
The Ku heterodimer, Ku70 and Ku80, plays a key role in DNA repair. Viable Ku70 knockouts exist in mice but not in human cell lines. The objective was to create Ku70 knockouts and evaluate knockout viability in human cells using CRISPR/Cas9 and TevCas9. Editing is achieved by Cas9 through one sequence-specific blunt cut accompanied by error-prone DNA repair. However, TevCas9, a novel fusion protein of Cas9 and I-Tev, creates two non-compatible DNA breaks and biases editing events towards a small deletion. Ku70 has five processed pseudogenes therefore intron-exon junctions were targeted by gRNA and a cell line stably transfected with an inducible second copy of Ku70-HA was used to compensate for the loss of endogenous Ku70. After transfection with Cas9 or TevCas9, monoclonal cell lines were picked. Analysis showed lowered or absent Ku70 expression. These Ku knockouts can be used to determine if Ku is required for human cell viability.
Summary for Lay Audience
The Ku protein, composed of two smaller proteins, Ku70 and Ku80, is important for maintaining the integrity of DNA, through its function in DNA repair. There may be additional functions for Ku in human cells as previous studies have reported that deleting Ku from human cells caused cell death suggesting that Ku is essential in human cells. The goal of this project is to generate a knockout cell that does not contain Ku70 to further study the role of Ku and to evaluate whether removing Ku70 will kill human cells or not. CRISPR/Cas9 is a popular tool used to remove a protein of interest from cells and gather knowledge on the protein by seeing the effect of removing it on the cell. TevCas9 is a variation of Cas9 that may be more effective and precise. We wanted to compare the efficiencies of Cas9 and TevCas9. Initially, trying to knockout Ku70 in immortalized human cells was unsuccessful. To test whether this meant Ku70 was essential or CRISPR-Cas9 was non-functional, we created a cell line with a second copy of the Ku70 that can be turned on and off when needed. If removing Ku70 is lethal, the second copy will produce some Ku70 protein that will keep the cell alive. This will allow the first copy to be safely removed by CRISPR without killing the cells. Subsequently, we will turn off the second copy too and observe whether cells can survive in the complete absence of Ku70. The generation of these cells will serve as the basis for studies investigating other roles of Ku outside well-known DNA repair and whether Ku has essential functions in human cells.
Parmar, Gursimran, "Generation of Conditional Ku70 Knockouts in Human Cell Lines Using CRISPR/Cas9 and Dual-Nuclease CRISPR/TevCas9" (2020). Electronic Thesis and Dissertation Repository. 6917.
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