A Well-Controlled BioID Design for Endogenous Bait Proteins

Abstract

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 Cas9cytidine 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.

Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector–Host Protein–Protein Interactions

“…An appropriate VLP control setup, i.e., a representation of the complete VLP proteome in absence of the effector, is indispensable for Virotrap MS-based analysis as VLPs exhibit their own characteristic proteome content [97]. In the context of appropriate BioID controls, it is noteworthy that by using the power of genome editing, Vandemoortele et al designed a method to obtain isogenic cell lines expressing T2A-BirA* bait fusion or its corresponding BirA* control protein, both under the control of the endogenous bait promoter [105]. The method relies on introduction of the T2A-BirA* cassette at the C-terminus of the protein of interest resulting in bicistronic expression of both the bait and BirA*, thereby serving an elegant control setup for isogenic cells obtained by targeting of a Cas9 enzyme, fused to a cytidine deaminase to the T2A autocleavage site, thereby inactivating the T2A peptide sequence resulting in the expression of the bait fusion from its endogenous promoter.…”

Nb-induced stabilisation of p53 in HPV-infected cells

“…Combination of this strategy with the Bio-ID proximity labelling would create a very powerful tool through which even weak and transient interactions could be identified. Interestingly, it has been recently demonstrated that this labelling strategy can be used for proximal protein mapping for endogenous p53 56 .…”

Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

“…For example, researchers can target a labeling enzyme to a different organelle or protein complex in addition to the primary target (Hung et al, 2014). Furthermore, isogenic cell lines can be used to avoid differences in transgene expression (Hesketh, Youn, Samavarchi‐Tehrani, Raught, & Gingras, 2017; Vandemoortele et al, 2019). (c) Background due to nonspecific labeling can be reduced by inducibly activating the labeling enzyme (Cho et al, 2017; Kehrer et al, 2016) or using endogenous CRISPR/Cas9 tagging of bait proteins to maintain physiological levels of the labeling enzyme (Long, Brown, & Sibley, 2018).…”