Hi,

I performed CRISPR-Cas9-mediated knock-in, to generate SOX2-(2X NLS)-tdTomato reporter hiPSC/hESC line (WTC-11 and H9).

Target locus is SOX2, before stop codon, following this article:

(Ref. Article Generation of a SOX2 reporter human induced pluripotent stem...

I obtained several colonies expressing tdTomato at the nuclei after puromycin (0.5 ug/ml) selection for 5-7 days, mechanically picked the colonies, and plated them into 96 well. After the colonies grew large enough, I passaged them with ReLeSR (Stem Cell Technologies) and plated them into 24 well culture plate. Again, colonies grown large in 24 well plate subsequently passaged into 6 well (also with ReLeSR), but problem happened at this point - spontaneously differentiated colonies suddenly increased! I've already tested whether mutations exist at the Left/Right homology arms at SOX2 locus, as well as other sites related to the off-target effects of gRNA, and found that all negative for mutations.

Does anyone experience similar situation and has any kinds of solutions??? T T