Endogenous tagging of intracellular structures with gene editing​

A. α-tubulin transfection
​​α​-tubulin gene-edited

B. Tom20 transfection
​Tom20 gene-edited

C. Desmoplakin transfection
​Desmoplakin gene-edited

Figure. Gene-editing versus transient transfection. Z-stacks of live hiPS cells comparing endogenous mEGFP tagging via gene-editing versus overexpression via transient transfection. All movies were imaged with a laser-scanning confocal microscope and images start from the bottom of the cells and end at the top. Movies shows the transient transfections and right panels the gene-edited cell lines for fluorescently tagged α-tubulin (A), desmoplakin (B) and Tom20 (C).​

Observations
Labeling structures with mEGFP endogenously (via CRISPR/cas9 gene editing) generates human cells that we can image with unprecedented clarity compared to tagged proteins that are generated by transient transfection. The extent of the difference can vary:

• Tagged α-tubulin: microtubules are more clearly visible due to decreased background when compared to overexpression
• Tagged desmomplakin: desmosomes localize to small puncta near the tops of cells. Transiently transfected cells form more and larger desmosomes or artificial desmosome-like structures.
• Tagged Tom20: overexpression of tagged Tom20 often generates sick cells with completely misshapen mitochondria compared to tagged Tom20 expressed at endogenous levels.