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  Targeted in situ protein diversification and intra-organelle validation in mammalian cells

Erdogan, M., Fabritius, A., Basquin, J., & Griesbeck, O. (2020). Targeted in situ protein diversification and intra-organelle validation in mammalian cells. Cell Chemical Biology, 27(5), 610-621.e5. doi:10.1016/j.chembiol.2020.02.004.

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 Creators:
Erdogan, Mutlu1, Author           
Fabritius, Arne1, Author           
Basquin, Jerome, Author
Griesbeck, Oliver1, Author           
Affiliations:
1Research Group: Tools for Bio-Imaging / Griesbeck, MPI of Neurobiology, Max Planck Society, ou_1113560              

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Free keywords: RED FLUORESCENT PROTEINS; HOMOLOGOUS RECOMBINATION; DIRECTED EVOLUTION; GENE; OLIGONUCLEOTIDES; ENDONUCLEASE; TECHNOLOGIES; CRISPR-CAS9; EXPRESSION; REPAIR
 Abstract: Engineered proteins must be phenotypically selected for function in the appropriate physiological context. Here, we present a versatile approach that allows generating panels of mammalian cells that express diversified heterologous protein libraries in the cytosol or subcellular compartments under stable conditions and in a single-variant-per-cell manner To this end we adapt CRISPR/Cas9 editing technology to diversify targeted stretches of a protein of interest in situ. We demonstrate the utility of the approach by in situ engineering and intra-lysosome specific selection of an extremely pH-resistant long Stokes shift red fluorescent protein variant. Tailoring properties to specific conditions of cellular sub-compartments or organelles of mammalian cells can be an important asset to optimize various proteins, protein-based tools, and biosensors for distinct functions.

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Language(s): eng - English
 Dates: 2020-05-21
 Publication Status: Published in print
 Pages: 17
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: Cell Chemical Biology
Source Genre: Journal
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Publ. Info: Cell Press
Pages: - Volume / Issue: 27 (5) Sequence Number: - Start / End Page: 610 - 621.e5 Identifier: ISSN: 2451-9456
CoNE: https://pure.mpg.de/cone/journals/resource/2451-9456