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

Cell Chemical Biology
Mutlu ErdoganOliver Griesbeck

Abstract

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.

References

Jun 21, 1994·Proceedings of the National Academy of Sciences of the United States of America·P RouetM Jasin
Aug 12, 2003·The Journal of Biological Chemistry·Jan PetersenJamie Rossjohn
Mar 3, 2004·Acta Crystallographica. Section D, Biological Crystallography·Laurent C StoroniRandy J Read
Dec 6, 2005·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Weimin LiuManfred Gossen
Feb 4, 2010·Acta Crystallographica. Section D, Biological Crystallography·Wolfgang Kabsch
Feb 4, 2010·Acta Crystallographica. Section D, Biological Crystallography·Paul D AdamsPeter H Zwart
Mar 10, 2010·Proceedings of the National Academy of Sciences of the United States of America·Kiryl D PiatkevichVladislav V Verkhusha
Apr 13, 2010·Acta Crystallographica. Section D, Biological Crystallography·P EmsleyK Cowtan
Aug 5, 2010·Journal of the American Chemical Society·Kiryl D PiatkevichVladislav V Verkhusha
Aug 10, 2010·Annual Review of Genetics·Wolf-Dietrich HeyerJie Liu
Aug 19, 2010·Nature Reviews. Genetics·Fyodor D UrnovPhilip D Gregory
Oct 23, 2010·Molecular Cell·Alberto Ciccia, Stephen J Elledge
Jan 13, 2011·Cell Cycle·Sandro ConradMarkus Löbrich
Apr 5, 2011·Acta Crystallographica. Section D, Biological Crystallography·Martyn D WinnKeith S Wilson
May 25, 2011·Methods in Molecular Biology·Richard J WardGraeme Milligan
Jul 19, 2011·Nature Methods·Fuqiang ChenGregory D Davis
Jan 14, 2012·Nucleic Acids Research·Yongwei ZhangWinfried Edelmann
May 9, 2012·Chemical Reviews·Fedor V Subach, Vladislav V Verkhusha
May 10, 2012·Immunologic Research·Shruti MaluPatricia Cortes
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Sep 11, 2012·Nature Methods·Amy J LamMichael Z Lin
Nov 22, 2012·Nature Reviews. Molecular Cell Biology·J Keith Joung, Jeffry D Sander
Mar 6, 2013·Frontiers in Molecular Neuroscience·Jasper AkerboomLoren L Looger
Jul 11, 2013·PloS One·Jie YangZhihong Zhang
Oct 26, 2013·Nature Protocols·F Ann RanFeng Zhang
Jun 10, 2015·Nature Reviews. Genetics·Michael S Packer, David R Liu
Jul 15, 2015·Nature Communications·Lindsey M CostantiniErik L Snapp
Aug 11, 2015·Nature Methods·Basil P HubbardDavid R Liu
Jan 13, 2016·Molecular Therapy : the Journal of the American Society of Gene Therapy·Morgan L Maeder, Charles A Gersbach
Apr 12, 2016·Biotechnology Advances·Patricia Molina-EspejaMiguel Alcalde
May 31, 2016·Nature Methods·Paula J CranfillDavid W Piston
Nov 9, 2016·Proceedings of the National Academy of Sciences of the United States of America·Fabrice GielenFlorian Hollfelder
Dec 31, 2016·International Journal of Molecular Sciences·Catherine StaudtMarielle Boonen
Jan 2, 2018·Cell Chemical Biology·Hajime ShinodaTakeharu Nagai
May 24, 2018·International Journal of Molecular Sciences·Hajime ShinodaTakeharu Nagai

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Citations

May 7, 2020·Nature Methods·Rita Strack
Oct 2, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Jasper Edgar NeggersDirk Daelemans
Jan 21, 2021·International Journal of Molecular Sciences·Brian Iaffaldano, Jakob Reiser
Mar 31, 2021·Current Opinion in Structural Biology·Oliver Griesbeck
Apr 9, 2021·Nature Methods·Samuel J Hendel, Matthew D Shoulders
May 23, 2020·Cell Chemical Biology·Evan M KerekBasil P Hubbard
Jun 10, 2021·Cell Chemical Biology·Cristina ChengJia Niu
Jun 12, 2021·Nature Communications·Mateusz MieczkowskiClaudia Höbartner
Jul 18, 2021·Science Advances·Xiao YiMichael Travisano
Jul 24, 2021·Chemical Reviews·Yajie WangHuimin Zhao

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