Dataset Information

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]

PMID: 30525648

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Project description:Recent optimization of CRISPR/Cas9-mediated genome engineering has resulted in the development of base editors that can efficiently mediate C>T and A>G transitions. Combining these genome engineering tools with human adult stem cell (ASC)-derived organoid technology holds promise for disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived hepatocyte, endometrial and intestinal organoids. First, using conventional and evolved Cas9-variants, we show efficacy of both cytosine and adenine base editors and use them to model four hot-spot point mutations in CTNNB1 in hepatocyte organoids. Next, we apply C>T base editors in endometrial organoids to insert nonsense mutations in PTEN and demonstrate tumorigenicity even in the heterozygous state. Furthermore, we use cytosine base editors for simultaneous oncogene activation (PIK3CA) and tumor-suppressor inactivation (APC and TP53). To increase the flexibility of base editor multiplexing, we then combine SpCas9 and SaCas9 base editors for simultaneous C>T and A>G editing at individual target sites. Finally, we show the power of base editor multiplexing by modeling colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAÃ¢Â€Â™s targeting four cancer genes. Seven clonal organoid lines and one bulk wild-type control sample were paired-end whole-genome sequenced using the Illumina Novaseq 6000 system. We sequenced four clonal intestinal organoid lines harbouring engineered TP53 and FBXW7 mutations as well as three lines targeted for oncogenic APC/TP53/PIK3CA/SMAD4 mutations. This WGS showed, as previously reported, a genome-wide increase in C>T mutations due to C>T base editor off-target activity, which is not enriched in predicted off-target regions based on the sgRNA sequences. Furthermore, we confirmed the absence of editing-induced driver mutations and lack of off-target mutational hotspots created by the genomic engineering.

Dataset Information

A well-controlled BioID design for endogenous bait proteins

Publications

A Well-Controlled BioID Design for Endogenous Bait Proteins.

Similar Datasets

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