When less is more - Endogenous tagging with TurboID increases the sensitivity of proximity labelling-based experiments

Abstract

AbstractIn recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Generally, protein fusions with labelling enzymes are transiently overexpressed to perform these experiments. Using a pipeline for the rapid generation CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. We found TurboID and its shorter variant miniTurboID to be superior above other labelling enzymes at physiological expression levels. Endogenous tagging of the μ subunit of the AP-1 complex increased the sensitivity for detection of interactors in a proximity labelling experiment and resulted in a more comprehensive mass spectrometry data set. We were able to identify several known interactors of the complex and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry data in just over a month.