Generation of Conditional Ku70 Knockouts in Human Cell Lines Using CRISPR/Cas9 and Dual-Nuclease CRISPR/TevCas9
Abstract
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.