Journal
CELL CHEMICAL BIOLOGY
Volume 27, Issue 5, Pages 610-+Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2020.02.004
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Funding
- Max Planck Society
- Human Frontiers Science Program
- Turkish Research Council TUBITAK [BIDEB-2219, 1059B191401130]
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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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