DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

CRISPR technology has made generation of gene knockouts widely achievable in cells. However,
once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present
DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon
combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon),
approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent
proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of
active gene expression with the ability to reactivate expression on demand, including activation of
silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and
we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking
regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live
microscopy from monoallelic modification, the potential to simultaneously image different alleles of
the same gene and bypasses the need to work with clones. Protein levels are easily tunable to
correspond with endogenous expression through cell sorting (DExCon), timing of light illumination
(LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach
allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor
recycling, expression kinetics and protein stability among highly similar endogenous Rab11 family
members and their colocalization in triple knock-in ovarian cancer cell lines.