## 4 Discussion
The latter proteins are essential for proper cell division in fish embryos, although their concerted function for centrosome structure remains unclear. Previous knockdown experiments in human cells indicated that acute knockdown of SSX2 IP weakens MT anchoring to centrosomes, leading to reduced delivery of protein material required for proper centriole assembly [9; 12]. In our experiments, we used human RPE-1 cells to establish homozygous KOs of _cep135_ and of its interaction partners WDR8 and SSX2 IP to analyze their functions in human somatic cells. All KOs produced viable and normally proliferating cells. KOs of _vdr8_ and _ssx2 ip_ showed no detectable defects in centrosome appearance and mitotic progression, despite centriolar satellites being more dispersed in both KO cell lines, possibly as a consequence of weakened MT anchorage at MTOCs. In turn, _cep135_ KO cells not only displayed dispersed centriolar satellites but showed diminished centrosonal targeting of \(\gamma\)-tubulin and CDK5 RAP2 as key molecular players of MTOC function. Previously published functions of both SSXZIP and WDR8 in somatic cells were mostly based on acute siRNA knockdown experiments in combination with rescues using exogenously expressed, siRNA-resistant variants [6; 8; 9; 10; 11; 12; 17]. CRISPR/Cas9-mediated KO approaches will select cells for compensatory actions that allow, if mechanistically possible, stable proliferation despite complete loss of function of individual gene products. Given that nonredundant, essential functions cannot be compensated for, we conclude that gene products of CEP135, WDR8, and SSX2 IP are not individually essential in human somatic cells in culture. This is in sharp contrast to the abovementioned RNAi experiments in the same (RPE-1) cells suggesting that loss of function of these genes forces cells to develop compensatory mechanisms that will finally allow regular proliferation of cells in culture. Compensation is most likely not possible, however, in developing vertebrates (medaka), where both knockdown and KO of _ssx2 ip_ and _Vdr8_ resulted in embryonic lethality after perturbed mitotic functions and chromosome segregation errors [6; 13]. Despite possible compensatory mechanisms in somatic KO cells, RPE-1 _cep135_ KOs displayed frequent centrosome cohesion defects, most likely caused by a general defect in PCM assembly. Diminished PCM in _cep135_ KO cells included reduced recruitment of the linker proteins C-NAP1 and rootletin. According to siRNA knockdown experiments in human cells, C-NAP1 has previously been shown to interact directly with CEP135 and to be required for its anchoring in the PCM [20; 30]. KO of _c-map1_ in human cells produced similar premature centrosome splitting to KO of _cep135_[31]. Our KO experiments, therefore, show that CEP135 is essential for interphase PCM assembly and that its function here cannot be compensated for. Human cells without CEP135 not only show loss of PCM and premature centrosome splitting, but also display a strong imbalance in PCM distribution between the split centrosomes. This results in defects of the interphase MT array that can be explained by misfunction of the MTOC. The overall reduced PCM and imbalance indicate that CCC is strongly compromised in _cep135_ KO cells. Although we did not further investigate MT-related functions in interphase, we consider it likely that _cep135_ KO cells show defects in Golgi organization and migratory behavior as previously observed for _c-map1_ KO cells [31]. Despite reduced and disbalanced interphase PCM, _cep135_ KO cells assemble mitotic PCM to a defined size at both centrosomes and progress through mitosis with balanced spindle poles, with symmetrical spindle structures, and without further delay. Not only do they show balanced distribution of \(\gamma\)-tubulin and CDK5 RAP2, but they also regain these PCM proteins to levels that even go beyond levels of these proteins on the spindle poles of mitosis in control RPE-1 cells.