A scalable strategy for high-throughput GFP tagging of endogenous human proteins.
A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼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.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143; Howard Hughes Medical Institute, University of California, San Francisco, CA 94143;
Manuel D
MD
Leonetti
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143.
Sayaka
S
Sekine
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 daichi.kamiyama@ucsf.edu jonathan.weissman@ucsf.edu bo.huang@ucsf.edu.
Daichi
D
Kamiyama
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143; Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; daichi.kamiyama@ucsf.edu jonathan.weissman@ucsf.edu bo.huang@ucsf.edu.
Jonathan S
JS
Weissman
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 daichi.kamiyama@ucsf.edu jonathan.weissman@ucsf.edu bo.huang@ucsf.edu.
Bo
B
Huang
D018274:Electroporation; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000235:genetics*
D011506:Proteins; D049452:Green Fluorescent Proteins
CRISPR/Cas9; GFP library; genome engineering