Noncanonical functions of Ku may underlie essentiality in human cells.

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.