Abstract
Kinetic simulation is a useful approach for the elucidation of complex cell-signaling systems. To perform the numerical simulations required for kinetic modeling, kinetic parameters such as the protein concentration and dissociation constant (Kd) are essential. However, only a limited number of kinetic parameters have been determined experimentally in living cells. Here, we describe a method for quantifying the concentration and dissociation constant (Kd) of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in (KI) and fluorescence cross-correlation spectroscopy (FCCS). First, the mEGFP gene was knocked-in at the end of the MAPK1 gene, which encoded ERK2, through homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ). Next, the HaloTag gene was further knocked-in at the end of the RSK2 gene. Protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag were quantified in living cells by fluorescence correlation spectroscopy (FCS), revealing substantial cellular heterogeneities. Interestingly, the levels of ERK2-mEGFP and RSK2-HaloTag were strongly positively correlated, suggesting a global mechanism underlying their expressions. In addition, FCCS measurement revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to EGF stimulation. Our method provides a new approach for quantification of the endogenous protein concentration and dissociation constant in living cells.