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Journal Article

Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells


Basquin,  Jerome
Conti, Elena / Structural Cell Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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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.

Cite as: https://hdl.handle.net/21.11116/0000-0006-9318-9
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.