Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 113, Issue 25, Pages E3501-E3508Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1606731113
Keywords
CRISPR/Cas9; GFP library; genome engineering
Categories
Funding
- NIH [R21MH101688]
- NIH Director's New Innovator Award [DP2OD008479]
- Howard Hughes Medical Institute
- Japan Society for the Promotion of Science Postdoctoral Fellowship for Overseas Researchers
Ask authors/readers for more resources
A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the similar to 20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome- wide analysis of protein localization and interaction networks in a native cellular context.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available