Proteomics

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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 overexpression issues. Control cell lines are typically provided by non-engineered cells or by engineered clones implying a considerable risk for artefacts because of clonal variation. Genome engineering can also be expected to transform BioID , an elegant proximity labeling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, which results in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins. To enable this design wherein, we introduced a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering. This leads to bi-cistronic expression of both p53 and BirA* under control of the endogenous promoter ensuring low background biotinylation in these control cells. By targeting a Cas9-cytidine deaminase base editor to the T2A auto-cleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. By A quantitative proteomics workflow we show significant benefits over other experimental settings including forced expression of p53-BirA* and parental control cell lines, and we provide a enabled a ffirst well-controlled view on the proximal proteins of endogenous p53. This novel application for base editors expands the CRISPR/Cas9 toolbox and may prove to be a valuable addition for synthetic biology.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Colon

SUBMITTER: Giel Vandemoortele  

LAB HEAD: Sven Eyckerman

PROVIDER: PXD011702 | Pride | 2019-06-17

REPOSITORIES: Pride

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Publications

A Well-Controlled BioID Design for Endogenous Bait Proteins.

Vandemoortele Giel G   De Sutter Delphine D   Moliere Aline A   Pauwels Jarne J   Gevaert Kris K   Eyckerman Sven S  

Journal of proteome research 20181210 1


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*, result  ...[more]

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