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Publication Abstract from PubMed

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.

Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells.,Erdogan M, Fabritius A, Basquin J, Griesbeck O Cell Chem Biol. 2020 Mar 3. pii: S2451-9456(20)30068-4. doi:, 10.1016/j.chembiol.2020.02.004. PMID:32142629^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.