1 Biology Department, Concordia University, Montreal, Canada.
2 Center for Microscopy and Cellular Imaging, Concordia University, Montreal, Canada.
3 Center for Applied Synthetic Biology, Concordia University, Montreal, Canada.

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG2_1-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG2₁₁). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.