A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Abstract

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. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2, 3). Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immunoprecipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism's proteome can be achieved. The power of systematic tagging approaches is best illustrated by studies conducted in the budding yeast Saccharomyces cerevisiae (4). In particular, a genome-wide collection of GFP-tagged yeast strains enabled the systematic study of protein localization in live cells (5), whereas libraries of strains expressing TAP epitope-fusion proteins paved the way for the large-scale isolation and proteomic analysis of protein complexes (6, 7). One of the great advantages of yeast genetics (especially in S. cerevisiae) is the efficiency and relative simplicity of PCR-based homologous recombination (8). As a result, functional tags can be easily inserted in a gene locus of interest, preserving endogenous expression levels and minimizing genomic disruption. Together, these genome-wide tagged libraries helped provide a comprehensive snapshot of the yeast protein landscape under near-native conditions (4, 5, 9-11).The development of clustered regularly interspersed short palindromic repeat associated protein 9 (CRISPR/Cas9)-based methods has profoundly transformed our ability to dir...

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

“…By fusing one fragment on a target protein and detecting its association with the other fragment, these constructs have demonstrated powerful applications in the visualization of subcellular protein localization [1][2][3] , quantification of protein aggregation 4 , detection of cytosolic peptide delivery 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3,9,10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .…”

“…With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP [1][2][3][4][5][6][7][8][9][10] in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly.…”

“…However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.…”

“…2 Protein labeling through transient expression or endogenous knock-in using mNG2 11 . a Fluorescence images of HeLa cells co-expressing either mNG2 11 or GFP 11 labeled H2B or CLTA with the corresponding FP [1][2][3][4][5][6][7][8][9][10] . Scale bars are 10 μm.…”

“…b FACS backgroud from wild-type HEK 293T cells, or HEK 293T cells stably expressing GFP [1][2][3][4][5][6][7][8][9][10] (using the stong SFFV promoter or the weak PGK promoter) or mNG2 [1][2][3][4][5][6][7][8][9][10] (using SFFV). c Comparison of tagging endogenous LMNA, RAB11A, and CLTA using mNG2 11 or GFP 11 knock-in in SFFV-mNG2 [1][2][3][4][5][6][7][8][9][10] and PGK-GFP 1-10 cells, respectively, (flow cytometry histograms) and the corresponding confocal images of mNG2 11 knock-in cells. Scale bars are 10 μm.…”