KCTD proteins have redundant functions in controlling cellular growth.

We explored functional redundancy of three structurally related KCTD proteins, KCTD2, KCTD5 and KCTD17 by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gb1 was involved in transcriptional control of gene expression, so we compared gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 lead to numerous changes in the expression levels of other G protein subunit genes while knockout of KCTD isoforms, had the opposite effect, presumably because of their role in regulating levels of Gb1. Our work demonstrates a unique relationship between KCTD proteins and Gb1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate. To explore the redundancy among KCTD proteins 2, 5, and 17 in the regulation of G-proteins and cellular growth, we conducted sequential CRISPR-KO targeting these proteins in HEK293 cells.