A Pilot Study On Regulatory Mechanisms Of CBX2 In Controlling Sexual Development Genes Using CRISPR/Cas9 Based Gene Editing System

CBX proteins play important roles in stem cell self-renewal and embryonic development as core components of mammalian Polycomb Repressive Complex PRC1.Knockout of mammalian CBX protein Cbx2 in mouse exhibit male-female sex reversal,similar to the reports of developmental abnormalities in human Cbx2 mutation cases.There is some experimental evidence suggesting that CBX2 regulates sex development by up-regulating the expression of Sry and Nr5a1 genes,which is inconsistent with the known repressive functions of PRC1,and the precise regulation mechanism is yet unclear.In order to further explore the roles of CBX2 in sex development,Cbx2 knockout,overexpression or other gene manipulations are necessary.As a new gene editing system,CRISPR/Cas9 has become a popular choice for mammalian gene manipulation.The benificial features such as simple,quick and efficient,attracted us to use the CRISPR/Cas9 system to carry out Cbx2 gene knockout.Through the establishment and continuous improvement of the CRISPR/Cas9 system,a high efficiency transient transfection experiment system and a lentiviral transfection system were developed,and a variety of cell lines capable of inducing expression of Cas9 were constructed,e.g.in human embryonic kidney cell line HEK293 T,mouse embryonic stem cell line E14TG2 a,mouse testicular teratoma cell line F9 etc.At present,homozygotic and heterozygotic Cbx2 KO HEK293 T cell lines have been established using our transient transfection system,and an experimental system has been established to verify the occurrence of knockout at either genomic sequence level or at protein level.In addition,we identified a new mutation(L4M)by sequencing the Cbx2 exons in the peripheral blood sample collected from a patient with sex developmental disorder(DSD).We also carried out some preliminary studies on a reporter system for the examination of CBX2 targeting specificity.This paper details the establishment of a variety of CRISPR/Cas9 experimental systems for gene knockout,and more importantly,the establishment of Cbx2 knockout cell lines may further reveal the roles of CBX2 in embryonic sex determination and differentiation,and whether its function relies on the canonical repressive function of PRC1.