Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2 alpha' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immunoprecipitates with the beta-subunit being downregulated by specific CK2 alpha' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2 alpha' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2 alpha/alpha'(-/-) cells relies on this deleted form of CK2 alpha', whose discovery provides novel perspectives about the biological role of CK2. (c) 2020 Elsevier Inc. All rights reserved.