Potential involvement of KANK1 haploinsufficiency in centrosome aberrations
KANK1 was found as a tumor suppressor gene based on frequent deletions in renal cell carcinoma and the inhibitory activity of tumor cell proliferation. Previously, we reported that knockdown of KANK1 induced centrosomal amplification, leading to abnormal cell division, through the hyperactivation of RhoA small GTPase. Here, we explored to the effect of loss of function of KANK1 through CRISPR/Cas9-based genome editing to knockout the gene. After several rounds of genome editing, however, there were no cell lines with complete loss of KANK1, and it was found that less the wild-type KANK1 dosage was, greater the number of cells with abnormal numbers of centrosomes and the rates of cell-doubling times and apoptosis were, suggesting the involvement of KANK1 haploinsufficiency in centrosome aberrations. RNA-sequencing analysis of the cells with reduced dosages of functional KANK1 revealed potential involvement of other cell proliferation-related genes, such as EGR1, MDGA2 and BMP3, which were reported to show haploinsufficiency when they function. Thus, haploinsufficiency of KANK1 may contribute to centrosome aberrations through the network of haploinsufficiency-related genes. Overall design: KANK1 partial knockout (partial KANK1-KO) cells were generated using the CRISPR/Cas9 system using guide RNA (gRNA) located immediate downstream of the first ATG. The duplex DNA with the sequences of 5'-TGACACCGGTGAGTTCAGAAGG-3', corresponding to the gRNA part, and its complementary 5'-CCTTCTGAACTCACCCGGGTGTCA-3', was incorporated into the pGuide-it-ZsGreen1 vector (Takara Bio, Shiga, Japan) to express Cas9 protein and gRNA. To introduce the vector into cells, 1.0 × 10^6 cells were pre-seeded and cultured at 37°C for 24 hr, and after the vector (1.2 µg) was introduced into the cells using a Lipofectamine 3000 Transfection kit (Thermo Fisher Scientific), the cells were cultured for 3 days.