Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells.
CRISPR/cas
directed evolution
fluorescent proteins
mammalian cell
organelle targeting
protein engineering
Journal
Cell chemical biology
ISSN: 2451-9448
Titre abrégé: Cell Chem Biol
Pays: United States
ID NLM: 101676030
Informations de publication
Date de publication:
21 05 2020
21 05 2020
Historique:
received:
29
07
2019
revised:
22
12
2019
accepted:
14
02
2020
pubmed:
7
3
2020
medline:
21
5
2021
entrez:
7
3
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32142629
pii: S2451-9456(20)30068-4
doi: 10.1016/j.chembiol.2020.02.004
pii:
doi:
Substances chimiques
Luminescent Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
610-621.e5Commentaires et corrections
Type : CommentIn
Type : CommentIn
Informations de copyright
Copyright © 2020 Elsevier Ltd. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Interests A patent application was filed relating to this work.