Mutational Analysis of a Transgenic Mouse Model of Human Sry

Testis development in males is controlled in most eutherian mammals, including humans, by the Y-chromosome gene SRY. Mutations of SRY result in differences/disorders of sex development (DSD), congenital conditions characterized by atypical development of the reproductive system. Thirty decades after its discovery, we still have an incomplete understanding of how this gene operates, and our translational efforts are dramatically hindered by the structural differences between mouse and human SRY. Most importantly, the lack of a system to experimentally generate mutations of human SRY and study their effect in vivo has limited progress in defining the mechanisms underlying the role of SRY in DSD. We have addressed this issue by generating a mouse model carrying a human SRY transgene able to drive male sex determination in XX mice. Using CRISPR-Cas9, we conducted a structure-function analysis of SRY by introducing novel modifications in vivo in each of its three domains (N-terminal, HMG box, and C-terminal), and characterizing the properties of the resulting variants. We showed that specific modifications resulted in complete or partial loss of SRY function, and that variation of the C-terminal - but not HMG box - domain affects protein stability rather than transcriptional activation capacity. Our results provide new functional insights unique to human SRY and the causes of DSD and present a versatile and powerful system for the study of SRY variations. This unique strategy has broad application potential, including in vivo functional studies of SRY variations, queries of the SRY mutation database, and the characterization of new pathogenic SRY variations found in clinical DSD.