A Well-Controlled BioID Design for Endogenous Bait Proteins

The CRISPR/Cas9 revolution
is profoundly changing the way life
sciences technologies are used. Many assays now rely on engineered
clonal cell lines to eliminate the overexpression of bait proteins.
Control cell lines are typically nonengineered cells or engineered
clones, implying a considerable risk for artifacts because of clonal
variation. Genome engineering can also transform BioID, a proximity
labeling method that relies on fusing a bait protein to a promiscuous
biotin ligase, BirA*, resulting in the tagging of vicinal proteins.
We here propose an innovative design to enable BioID for endogenous
proteins wherein we introduce a T2A-BirA* module at the C-terminus
of endogenous p53 by genome engineering, leading to bicistronic expression
of both p53 and BirA* under control of the endogenous promoter. By
targeting a Cas9-cytidine deaminase base editor to the T2A autocleavage
site, we can efficiently derive an isogenic population expressing
a functional p53-BirA* fusion protein. Using quantitative proteomics
we show significant benefits over the classical ectopic expression
of p53-BirA*, and we provide a first well-controlled view of the proximal
proteins of endogenous p53 in colon carcinoma cells. This novel application
for base editors expands the CRISPR/Cas9 toolbox and can be a valuable
addition for synthetic biology.