Intersex conditions are a spectrum of conditions in which gonadal, chromosomal, or anatomical sex is atypical. The underlying molecular basis remains unknown in many forms of DSD. This is due, in part, to the lack of manipulable models that recapitulate human sex determination. Sex is determined by the onset of SRY expression around gestational week 6 in the XY embryo. In turn, the transcription factor SRY switches on expression of SOX9, another DSD gene. SOX9 is a critical hub gene that regulates dozens of target genes we seek to identify, as they may contribute to DSD etiology. Towards this, we characterised NT2/D1 cells as an in vitro model to model Sertoli cell function in the testis. NT2 cells are a human multipotent clonal cell line derived from a testicular tumour that model a variety of human developmental processes. Undifferentiated, NT2 model sex determination initiated by SRY or by SOX9. When differentiated, NT2 model neuronal development or smooth muscle development. However, as a multipotent line, NT2/D1 cells are difficult to manipulate e.g. by RNAi. To extend the utility of NT2/D1 cells, we have established an NT2/D1-cas9 cell line. We characterise the integrity of these via a suite of cell phenotyping assays including xCELLigence. We are using NT2/D1-cas9 to knockout candidate SOX9 target genes, and investigating the cellular behaviours affected in DSD: cell adhesion, proliferation and migration.