It is unclear why TagRFP expression does not fully recapitulate endogenous shha mRNA expression. We currently speculate that this is attributable to inefficient cleavage of the 2A peptide sequence. A glycine-serine-glycine spacer was recently revealed to significantly improve the cleavage efficiency of the 2A peptide (Wang Y, et al. Scientific Report2015), but we were unable to use this spacer because it was reported after the construction of Zwitch. We will address this issue in a future study to improve the visualization of mutant cells using Zwitch.