The Fragile-X related disorders (FXDs) are Repeat Expansion Diseases (REDs) that 
result from expansion of a CGG-repeat tract located at the 5' end of the FMR1 
gene. While expansion affects transmission risk and can also affect disease risk 
and severity, the underlying molecular mechanism responsible is unknown. Despite 
the fact that expanded alleles can be seen both in humans and mouse models in 
vivo, existing patient-derived cells do not show significant repeat expansions 
even after extended periods in culture. In order to develop a good tissue 
culture model for studying expansions we tested whether mouse embryonic stem 
cells (mESCs) carrying an expanded CGG repeat tract in the endogenous Fmr1 gene 
are permissive for expansion. We show here that these mESCs have a very high 
frequency of expansion that allows changes in the repeat number to be seen 
within a matter of days. CRISPR-Cas9 gene editing of these cells suggests that 
this may be due in part to the fact that non-homologous end-joining (NHEJ), 
which is able to protect against expansions in some cell types, is not effective 
in mESCs. CRISPR-Cas9 gene editing also shows that these expansions are 
MSH2-dependent, consistent with those seen in vivo. While comparable human 
Genome Wide Association (GWA) studies are not available for the FXDs, such 
studies have implicated MSH2 in expansion in other REDs. The shared unusual 
requirement for MSH2 for this type of microsatellite instability suggests that 
this new cell-based system is relevant for understanding the mechanism 
responsible for this peculiar type of mutation in humans. The high frequency of 
expansions and the ease of gene editing these cells should expedite the 
identification of factors that affect expansion risk. Additionally, we found 
that, as with cells from human premutation (PM) carriers, these cell lines have 
elevated mitochondrial copy numbers and Fmr1 hyperexpression, that we show here 
is O2-sensitive. Thus, this new stem cell model should facilitate studies of 
both repeat expansion and the consequences of expansion during early embryonic 
development.