Genetic code expansion for temporal controlled labeling of endogenous expressed proteins

Temporal labeling of proteins is a central requisite for studying protein lifetimes, trafficking, and abundance changes in a cellular context. In this talk I will present how to combine genetic code expansion and CRISPR/Cas9 genome engineering to label specifically endogenously expressed proteins with high spatio-temporal resolution. The developed method, named SCROL (Stop-Codon-Read-thrOugh-Label), exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with unnatural amino acids (UAAs) to create stop codon read through events and expand protein sequences with a label. Upon timely controlled addition of a UAA, the protein sequence can be extended with a label of choice. We demonstrated the functionality of SCROL by pulse labeling of endogenous b-actin and p53 with a minimally invasive HA tag at their C-terminal ends. Synthesis rates and subcellular localization of the proteins were quantified at the single cell level with a proximity ligation assay. In contrast to existing technology, SCROL maintains the endogenous transcriptional regulatory background of the cell and does not perturb protein function in absence of the UAA.