ausblenden:
Schlagwörter:
RED FLUORESCENT PROTEINS; HOMOLOGOUS RECOMBINATION; DIRECTED EVOLUTION;
GENE; OLIGONUCLEOTIDES; ENDONUCLEASE; TECHNOLOGIES; CRISPR-CAS9;
EXPRESSION; REPAIR
Zusammenfassung:
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.
