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

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Erdogan M., Fabritius A., Basquin J., Griesbeck O.

CELL CHEMICAL BIOLOGY, cilt.27, sa.5, ss.610-626, 2020 (SCI-Expanded, Scopus) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 27 Sayı: 5
  • Basım Tarihi: 2020
  • Doi Numarası: 10.1016/j.chembiol.2020.02.004
  • Dergi Adı: CELL CHEMICAL BIOLOGY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, EMBASE, MEDLINE, Veterinary Science Database
  • Sayfa Sayıları: ss.610-626
  • Açık Arşiv Koleksiyonu: AVESİS Açık Erişim Koleksiyonu
  • Ankara Üniversitesi Adresli: Hayır

Özet

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.