CRISPR-Mediated Tagging of Endogenous Proteins with a

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CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide Marie K. Schwinn, Thomas Machleidt, Kris Zimmerman, Christopher T. Eggers, Andrew S. Dixon, Robin Hurst, Mary P. Hall, Lance P Encell, Brock F. Binkowski, and Keith V. Wood ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00549 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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ACS Chemical Biology

CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide Marie K. Schwinn*,†,§, Thomas Machleidt*,†, Kris Zimmerman†, Christopher T. Eggers†, Andrew S. Dixon‡, Robin Hurst†, Mary P. Hall†, Lance P. Encell†, Brock F. Binkowski†, and Keith V. Wood† †

Promega Corporation, Madison, Wisconsin 53711 United States Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112 United States ‡

ABSTRACT Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

INTRODUCTION As protein expression and post-translational modifications are fundamental to cellular physiology, their disruption can lead to pathophysiological conditions such as cancer, neurodegeneration, and inflammatory diseases.1, 2 Elucidating the intrinsic dynamics of these processes can thus offer insight into disease mechanisms and provide predictive models for drug discovery. Common approaches for quantifying protein levels and their modifications in complex biological samples include mass spectrometry and antibody-based methodologies, such as ELISA and Western blotting. Mass spectrometry is relatively low-throughput and usually requires specialized procedures to achieve reliable quantitation. It also has limited dynamic range and struggles with detection of low abundance proteins.3 While antibody detection systems share many of these limitations, dependable quantitation is also limited by the availability of high-quality antibodies.4 Dynamic processes in cells are routinely revealed using genetically encoded protein fusions with reporter tags, most commonly GFP or similar autofluorescent proteins. Typically, this is accomplished in mammalian cells by overexpressing recombinant proteins from genetic vectors containing strong transcriptional promoters. However, by

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omitting the endogenous genetic loci for protein expression, the absence of proper regulatory elements can produce an imbalance in protein levels leading to non-physiological artifacts.5 In recent years, CRISPR/Cas9 has emerged as a prominent technology for genome editing due to its simplicity and ease of use. By combining the site-specific cleaving abilities of CRISPR/Cas9 with the native cellular DNA repair mechanisms, reporters can be precisely inserted at endogenous loci. CRISRP/Cas9-mediated integration of full-length GFP and luciferase reporters has been reported, although the procedures are generally cumbersome and relatively inefficient.6, 7 Recently, the ability to tag endogenous proteins with a 16-amino acid peptide derived from GFP was described.8 This peptide tag (GFP11) generates fluorescence when co-expressed with its complementary fragment, GFP1–10. The small size of the tag allowed the donor DNA templates encoding GFP11 to be made as a synthetic single-stranded oligodeoxynucleotides (ssODNs). In combination with a ribonucleoprotein complex (RNP) comprising synthetic guide RNA (gRNA) and purified Cas9, this allowed efficient genome editing without molecular cloning. While the productivity of this approach encourages proteome-wide tagging, the detection sensitivity of GFP may be insufficient for the expression level of most endogenous proteins.8 In particular, proteins involved in cellular signaling tend to be expressed at relatively low levels and thus may be difficult to detect. Moreover, fluorophore maturation of the reconstituted GFP complex is quite slow, limiting its suitability for quantifying dynamic processes.9 Conversely, luciferase reporters are recognized for providing highly sensitive detection and rapid quantitation over an extensive concentration range.10 Taking advantage of this, we independently developed a similar approach for tagging endogenous proteins based on the very bright NanoLuc luciferase. This luciferase is approximately 100-fold brighter than firefly or Renilla luciferases, and its molecular weight (19 kDa) is substantially less than GFP.11 Complementing subunits of a split NanoLuc were previously engineered to accurately measure protein interaction dynamics within cells.12 This reporter system (NanoBiT) comprises an 11-amino acid peptide (SmBiT) that interacts with very low affinity (KD > 100 µM) to an 18 kDa polypeptide (LgBiT) to form a luminescent complex. During the development of this system, other peptides of different affinities to LgBiT were also identified. One in particular (HiBiT) was found to have an exceptionally high affinity (KD = 700 pM) to LgBiT. Through its ability to efficiently complex with LgBiT, we envision that HiBiT could serve as a quantitative luminescent peptide tag. Due to its small size, we also reasoned that HiBiT is well-suited for efficiently tagging endogenous proteins using ssODN donor templates together with CRISPR/Cas9 RNP complexes. To evaluate HiBiT as a means for quantifying cellular protein levels, we selected the hypoxia-inducible pathway in which both protein abundance and post-translational modifications play a role in regulating cellular functions.13 The hypoxia-inducible factor 1A (HIF1α) regulates many cellular processes, including angiogenesis, cell survival, migration, and nutrient metabolism. Altered expression of HIF1α can trigger aberrant activation of downstream genes, potentially contributing to one of several HIF1αassociated pathologies. We show that appending HiBiT onto HIF1α and associated proteins can be accomplished rapidly using Cas9 RNP complexes and ssODN homology-directed repair (HDR) templates. The high integration efficiency and assay sensitivity of HiBiT enable quantification of protein levels in the mixed population of edited cells without requiring clonal isolation. This allows reporter measurements to be made within 24 to 48 h of editing in either live cells or lysates.


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In addition to reporting on protein expression, the HiBiT tag also creates an avenue for detecting covalent modifications on the tagged protein. A myriad of post-translational modifications mediate diverse protein attributes, including conformation, processing, localization, activity, and interactions.14 Immunoassays provide a convenient means for detecting these modifications, although achieving selectivity for a single target can be challenging. With this in mind, we developed a homogenous immunoassay for measuring post-translational modifications that relies upon bioluminescence resonance energy transfer (BRET) between the HiBiT/LgBiT reconstituted luciferase and a fluorescentlylabeled antibody. Because BRET data is presented as a ratio of signals from the energy donor (i.e., the luciferase) and acceptor (i.e., the fluorophore), this approach indicates the degree of modification normalized to protein abundance. Detection specificity is achieved because antibody bound to inappropriate targets is non-productive for energy transfer due to absence of donor signal. Dynamic regulation of these modifications can also be correlated to changes in protein expression, since the energy donor simultaneously provides a measure of protein abundance. We used this to correlate prolyl hydroxylation of HIF1α with the controlled degradation of this transcription factor. The coordinated fluctuations revealed by this method corroborate the underlying mechanism linking these elements in the signaling pathway. Even though a fluorophore is involved in this assay design, high detection sensitivity and dynamic range are achieved due to the bright luminescence of the energy donor and large spectral shift to the acceptor (>175 nm).15 Correlations involving post-translational modifications should be possible simply by changing the binding specificity of the fluorescent antibody. Together, our results demonstrate that the HiBiT peptide tag can readily be appended onto endogenous proteins using CRISPR/Cas9, allowing sensitive quantification of both protein abundance and posttranslational modifications.

RESULTS AND DISCUSSION HiBiT Quantitation. For quantifying protein abundance, it is essential that the luminescence of HiBiT be proportional to its concentration when reconstituted with the LgBiT. Moreover, to be suitable across the broad range of expression levels exhibited by endogenous proteins, this linear relationship must extend to very low concentrations. For both full-length NanoLuc and LgBiT complexed with the low affinity SmBiT, this has been demonstrated previously through biochemical means, as well as in living and lysed cells.11, 12 Using purified proteins in a biochemical format allows unambiguous correlation of luminescence to the concentration of the tag. A serial dilution of purified HiBiT fusion in the presence of saturating LgBiT produced a linear correlation extending over 8 orders of magnitude, with similar results obtained both in buffer and in the presence of cell lysates (Figures 1a, s1). Light intensity was also comparable to NanoLuc, with measurable signal detected to less than 1 amol. In a typical sample of 10,000 cells, this would correspond to about 10 molecules per cell. Titration of transfected DNA encoding HiBiT into cultured mammalian cells produced a linear correlation spanning 4 orders of magnitude, evident for both live cells and cell lysates (Figure 1b). Measurement of HiBiT in live cells was achieved by having the LgBiT expressed from a chromosomally integrated expression cassette. As the non-linearity evident at high DNA concentration is similar for both lytic and live cells, this is apparently not caused by any restriction in measuring the HiBiT/LgBiT complex within the cells. Considering that detection of HiBiT



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from lysates should be analogous to the biochemical format, the comparably reduced linear range found using cells is probably caused by a limitation of protein expression from transfected DNA. HiBiT Tagging of HIF1α α. To append HiBiT onto endogenous proteins, we electroporated RNP complexes consisting of recombinant Cas9 and synthetic gRNA into cells in the presence of ssODN donor template (Figure s2).8 To minimize introduction of insertions or deletions (indels) within gene coding sequences, gRNA sequences were designed, when possible, to direct Cas9 cleavage to untranslated regions, while donor templates were designed to prevent re-cutting of edited genes by disrupting the gRNA target sequence (Table s1). Expression of HiBiT in the edited cell pools was readily confirmed by measuring luminescence in lysates following addition of recombinant LgBiT. Because expression of the peptide tag directly from a ssODN template is highly improbable, as is a random integration resulting in expression of a functional peptide, luminescence in the presence of LgBiT signifies HiBiT expression at the targeted loci. We assessed the effectiveness of this approach by editing HeLa cells to express HIF1α with HiBiT fused to the C-terminus. The frequency of HiBiT insertion across all alleles in the pools of edited cells was approximated by quantitative PCR using primers designed to recognize the sequences encoding either HIF1α-HiBiT or total HIF1α. From five independently generated pools of edited cells, the average allelic editing efficiency was determined to be 52 ± 16% (Table s2). Clonal cell populations were isolated by limiting dilution and used for further genetic characterization of the edited cells. Targeted amplicon sequencing of genomic DNA isolated from clones confirmed the presence of in-frame HiBiT insertion. The dynamic regulation of HIF1α expression offered an apt model for assessing rapid changes in protein abundance. Under normal oxygen conditions, HIF1α is rapidly turned over via a mechanism that involves HIF1α hydroxylation by prolyl hydroxylase-domain (PHD) enzymes, recognition and ubiquitination by the E3 ligase von Hippel Lindau (VHL) tumor suppressor protein, and degradation by the proteasome. Conversely, oxygen deprivation leads to inhibition of PHD enzymes, loss of hydroxylation of proline residues 402 and 564, and stabilization of HIF1α.16 As expected, luminescence corresponding to HIF1α-HiBiT was low, but still above background, when edited cells were grown under normoxic conditions. However, the luminescent signal increased over 7-fold after transferring the cells to a hypoxia chamber for 6 h, reflecting stabilization of the HIF1α-HiBiT (Figure 2a, s3a–b). The signal correlated with the accumulation of total HIF1α protein in the edited cells, as determined by Western blotting (Figure s3c–d). Modulation of HIF1α levels can also be achieved pharmacologically with inhibitors of prolyl hydroxylases (phenanthroline, ML228, DFO, FG2216, FG4592, DMOG, and IOX2), the proteasome (MG132), and the NEDD8-activating enzyme (MLN4924).17-22 Treatment of cells with these compounds revealed the predicted increase in luminescence in the HIF1α-HiBiT edited cells but not unedited HeLa cells (Figure 2b, s4a–b, Table s3). While these data were collected using pools of edited cells, equivalent results were obtained for clonal populations (Figure s4c). To verify that the luminescence originates from the correct HiBiT-tagged protein, an antibody-free blotting technique was used to confirm a single luminescent band corresponding to the correct molecular weight (Figure s4d). Stabilization and proper nuclear localization of the HiBiT-tagged HIF1α was also observed by luminescence imaging of live cells after phenanthroline treatment (Figure s5). By increasing exposure time during imaging, HIF1α-HiBiT was also detectable in the cytoplasm of cells grown under normal oxygen conditions, as previously reported.23


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The varied responses to different compounds suggests that HIF1α-HiBiT could be used to quantify drug potency and efficacy. Measurement of HIF1α-HiBiT luminescence from cells treated with dilutions of phenanthroline, MG132, and MLN4924 revealed both differing potencies (5.4 µM, 4.4 µM, and 350 nM, respectively) and maximal efficacies (Figure 2c). Western blotting for total HIF1α confirmed that the dose-dependent increase in luminescence was accompanied by a corresponding increase in protein levels (Figure s6a–b). Tagging Downstream Targets. Because HIF1α is a key regulator of cellular adaptation to hypoxia, significant effort has been centered on identifying drugs that modulate HIF1α and its downstream effects. However, the mechanism by which cells respond to hypoxia is complex and involves transactivation of numerous genes responsible for cell survival. How these downstream regulators contribute to malignancy remains unclear, but understanding their mode of action could aid in the identification of disease markers and future therapeutic targets. We selected four putative HIF1α target genes (ANKRD37, BNIP3, HILPDA, and KLF10) identified in previous studies to tag with HiBiT.24 The allelic HiBiT editing efficiency at these loci ranged from 6 to 80% (Table s4). Differences in editing efficiency for different genes could simply be attributed to gRNA and HDR template design; however, it could also reflect the chromatin state at the individual editing sites.25 Targeted amplicon sequencing of the insertions in clonal populations confirmed in-frame HiBiT insertion at the c-terminus of the genes. With HiBiT appended onto these genes, the regulated expression of their corresponding proteins could be examined under hypoxic conditions. Expression of ANKRD37, BNIP3, and HILPDA increased by 6-, 12, and 3-fold, respectively, indicating that these targets are upregulated under hypoxic conditions (Figure 3a–c, s7a–c). Interestingly, no significant elevation in KLF10 protein was observed, suggesting that KLF10 is not directly involved in the immediate hypoxic response (Figure 3d, s7d). The observed differences in signal intensity among the various targets may arise from several possible factors, including variations in editing efficiencies, gene expression, or protein stabilities. Induction of protein expression was also examined with the same inhibitor panel used to induce HIF1α levels. Only BNIP3 responded similarly to the compounds as HIF1α, while ANKRD37 and HILPDA exhibited activation profiles markedly distinct from HIF1α (Figure 4a–c, s8a-c, Table s5). These results suggest that up-regulation of ANKRD37 and HILPDA may involve additional factors in the hypoxia response. In contrast to the other targets, KLF10 showed no appreciable increase in protein abundance following treatment with PHD inhibitors, implying that KLF10 is not a direct target of HIF1α (Figure 4d, s8d). However, the proteasome inhibitor MG132 stimulated a 4-fold increase in KLF10, indicating that protein turnover may be involved in regulation of KLF10 levels. The findings obtained in these single dose experiments were further corroborated in compound titration experiments (Figure 5a–d, Table s6). Dose-response curves generated from the edited cells show markedly distinct compound profiles, further supporting that the mechanism by which expression of these proteins is regulated involves factors in addition to HIF1α. Again, the compound profile of BNIP3 most closely matches that of HIF1α. In addition, phenanthroline-inducible expression and proper subcellular localization of the ANKRD37, BNIP3, HILPDA, and KLF10 were confirmed using bioluminescence imaging (Figure s9).



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HiBiT Tagging in Primary Cells. Although CRISPR/Cas9-mediated HiBiT tagging was an effective strategy for detecting endogenous proteins in HeLa cells, applying this method in primary cell lines could potentially present additional challenges. Primary cells typically resemble the differentiated phenotype of the tissues from which they were derived and are believed to provide a more biologically relevant model for studying certain aspects of cellular physiology. However, use of these cells can be complicated by more complex culturing procedures, lower transfection efficiencies, and finite cell divisions. These same limitations may complicate the ability to utilize CRISPR/Cas9 for inserting reporter tags. Nonetheless, considering the high detection sensitivity achievable with HiBiT, CRISPR-mediated tagging may be sufficiently efficient in primary cells to support luminescent measurements directly in the unsorted pools. To evaluate the feasibility of CRISPR-mediated tagging in primary cells, we attempted to tag HIF1α, the putative HIF1α target BNIP3, and the angiogenic growth factor VEGFA using primary human umbilical vein endothelial cells (HUVECs). Pools of edited cells were treated with a subset of the compounds we had used to induce HIF1α expression. Significant increases in luminescence of at least 2-fold were detectable following incubation with the PHD enzyme inhibitors (phenanthroline, ML228, and DFO) but not the proteasome or neddylation inhibitors (Figure 6a–c, Table s7). The magnitude of induction was lower in HUVEC cells compared with the results obtained in HeLa cells. However, the results are consistent with published results and may reflect differing genetic backgrounds between the cell types.26 Real-Time Measurements in Cells. Because cells are programmed to react rapidly to environmental changes, the ability of a reporter to respond to variation without delay becomes crucial for the analysis of dynamic processes within the cell. In particular, being able to measure protein abundance in real time provides temporal information that is exceedingly difficult to obtain using endpoint assays. In the case of hypoxia, cells must quickly adapt to oxygen deprivation by stimulating pathways that lead to HIF1α stabilization and subsequent activation of survival genes. We wanted to determine if HiBiT could provide a real time readout of temporal changes in endogenous protein levels in the HIF1α pathway. To analyze expression kinetics in living cells, clonal cell lines having HiBiT tagged onto HIF1α, BNIP3, HILPDA, ANKRD37, and KLF10 were transduced with BacMam containing an expression cassette for LgBiT, and luminescence was recorded for 10 h following phenanthroline treatment (Figure 7a). HIF1α accumulation was detected within 30 min of compound addition, reached a steady state after 3 h, and remained steady for the duration of the experiment. This was also observed using luminescence imaging (Figure s10). Increases in the HIF1α-inducible proteins (BNIP3, HILPDA, and ANKRD37) were not detectable until the HIF1α reached a steady state. Interestingly, accumulation of the target proteins appears to occur in a sequential fashion with ANKRD37 emerging soon after HIF1α induction. In contrast, accumulation of HILPDA and BNIP3 is delayed by as much as 3 h compared to ANKRD37. Consistent with previous results, KLF10 did not show appreciable elevation over the tested time period. Reoxygenation of hypoxic tissues or cells leads to rapid reactivation of PHD enzymes resulting in hydroxylation and degradation of HIF1α.27 Previous studies used Western blotting to show that this process was rapid with an estimated half-life for HIF1α of less than 10 min after exposure to normoxic conditions. We were interested if it was possible to measure degradation of HIF1α after reoxygenation in real time. Following incubation of HIF1α-HiBiT edited cells in a hypoxia chamber, cells were returned


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to normoxia, and luminescence was measured every 30 s. The results show a brief plateau followed by rapid decline of signal indicating a half-life for HIF1α-HiBiT of less than 6 min, which is in agreement with previously published data (Figure 7b).28 Measuring Post-Translational Modifications. Post-translational modifications often serve as the mechanistic triggers in cell signaling. Regarding hypoxia, both activity and stability of HIF1α are intimately connected to several covalent post-translational modifications, including hydroxylation, acetylation, and phosphorylation.29 For example, hydroxylation of HIF1α at proline residues 402 and 564 promotes interaction of HIF1α with VHL, followed by ubiquitination, and ultimately degradation. Recognizing that immunoassays can present a convenient and inexpensive approach for detecting protein modifications, we devised a homogenous format that relies upon bioluminescence resonance energy transfer (BRET) between the HiBiT/LgBiT luciferase complex and a fluorophore-labeled antibody. The energy transfer is proportional to the amount of modification of interest normalized to the tagged target protein. To detect changes in hydroxylation at proline residue 564 (hydroxy-Pro564) upon inhibition of PHD enzymes, edited cells were treated with a serial dilution of phenanthroline combined with a proteasome inhibitor (MG132) to slow the degradation of HIF1α. Detection of hydroxy-Pro564 HIF1α was performed by first replacing the medium with lysis buffer containing recombinant LgBiT and a hydroxyPro564 HIF1α antibody. Following addition of fluorescently labeled secondary antibody and furimazine, BRET signal was recorded by measuring luminescence in the donor (460 nm) and acceptor (600 nm) channels. As anticipated, increasing concentrations of phenanthroline led to a decrease of the BRET ratio, which is indicative of declining hydroxylation at Pro564 (Figure 8). The decline of prolyl hydroxylation should lead to increased levels of HIF1α. Indeed, increased luminescence from HiBiT was observed, and it positively correlated with phenanthroline concentration. This correlation was also confirmed by Western blotting (Figure s11). The mechanistic linkage between prolyl hydroxylation and HIF1α concentration is further corroborated by closely matching EC50 values of 1.7 and 2.8 µM, respectively. We envision that this approach will provide a simple, robust, and scalable means for quantifying the dynamics of post-translational modifications associated with HiBiT-tagged endogenous proteins. Summary. By combining CRISPR/Cas9 editing and HiBiT reporter technologies, we have developed a simple approach for tagging endogenous proteins with a small luminescent peptide. The HiBiT tag can be rapidly assayed with high sensitivity and linearity, allowing quantitative assessment of the dynamic processes associated with cellular signaling. This report focuses on capabilities for determining changes in protein expression and post-translational modification, although assay configurations for other processes can be contemplated. Tagging endogenous proteins with HiBiT is achieved without molecular cloning simply by delivering synthetic gRNA and oligonucleotides with purified Cas9 into the cell. Owing to the rapid action of the Cas9 nuclease and the native cellular repair processes, expression of the tagged proteins can be measured within 24–48 h of editing. Thus, this method enables the analysis of cellular proteins with the ease and speed of a transient transfection experiment but without the potential artifacts of ectopic expression. CRISPR-mediated HiBiT tagging of endogenous proteins is a scalable strategy for investigating protein abundance and modifications that circumvents shortcomings of conventional methodologies, most



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notably immunoassays. Despite the longstanding popularity of immunoassays for investigating signaling pathways, obtaining reliably quantitative data through this means remains technically challenging. Central to the success of these assays is the ability to acquire a high-quality, specific antibody for the protein of interest. Although we were able to locate a qualified antibody for specific detection of HIF1α, availability of antibodies for the other target proteins was limited and protocols for their use in blotting were not as thoroughly established. In contrast, we were able to append HiBiT onto all of our desired target proteins and subsequently quantify their abundance in response to various stimuli. Unreliable antibody specificity can also be problematic in the detection of post-translational modifications. Our BRET immunoassay resolves this, as only proteins tagged with HiBiT can generate a signal indicative of antibody binding. While antibody specificity is often limiting, low sensitivity can also restrict the ability to obtain quantitative data for many proteins. In order to capture low abundance proteins, processing of large quantities of sample may be necessary. Assay sensitivity for HiBiT allows sub-attomole detection, which should be sufficient for measuring most cellular proteins. Indeed, we were able to detect each of our target proteins without enrichment steps in a multi-well assay format. Significantly, this could provide a means for screening a library of compounds to identify those able to modulate specific signaling pathways. A key differentiating aspect of HiBiT is the ability to quantify expression dynamics in real time. By introducing the complementing LgBiT subunit into cells (e.g., by viral delivery or plasmid transfection), changes in protein abundance can be observed within living cells. This can provide temporal information that is difficult to obtain from endpoint assays. Using HiBiT-edited cell lines transduced with BacMam-LgBiT, we were able to measure temporal changes of protein accumulation and degradation of HIF1α and its putative target proteins under different physiological conditions. Ultimately, we believe that the HiBiT peptide tag will provide a broadly beneficial capability for investigating protein dynamics in an appropriate physiological context.

METHODS Cas9, gRNA, and ssODN. Prior to experimentation, recombinant Streptococcus pyogenes Cas9 tagged on the N-terminus with a histidine-nuclear localization signal-myc tag (Aldevron) was diluted to 20 µM in Cas9 buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM TCEP). gRNA were assembled by incubating 1 nmol Alt-R CRISPR RNA (crRNA) with 1 nmol Alt-R trans-activating crRNA (tracrRNA) in 50 µl NucleaseFree Duplex Buffer (Integrated DNA Technologies, IDT) at 95 oC for 5 min and then cooling to ambient temperature. ssODN donor DNA templates were purchased as single-stranded Ultramer DNA Oligonucleotides (IDT) and resuspended to 100 µM in nuclease-free water. RNP Complex Assembly and Delivery. RNP complexes were assembled and electroporated into cells, as previously described.30 Briefly, 100 pmol Cas9 and 120 pmol gRNA were incubated for 10 min at ambient temperature. Cells (2 x 105) were resuspended in 20 µl 4D Nucleofector solution SE for HeLa or SF for HUVEC cells (Lonza). RNP complex and 100 pmol of donor DNA were then electroporated into the cells with the 4D Nucleofector System (Lonza) using program CN-114 for HeLa and DN-100 for HUVEC.


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Following electroporation, cells were incubated at ambient temperature for 5 min and then transferred to a 6-well plate for culturing. 24–48 h post-electroporation, cells were analyzed for insertion. Lytic HiBiT Detection. Edited cells were resuspended to 2 x 104 cells in 100 µl growth medium, plated in solid white 96-well tissue culture plates (Corning Costar #3917), and cultured for 24 h prior to treatment. Cells were then exposed either to modulators of the hypoxia pathway or to hypoxia for 6-24 h. An equal volume of Nano-Glo HiBiT Lytic Reagent (Promega N3030), consisting of Nano-Glo HiBiT Lytic Buffer, Nano-Glo HiBiT Lytic Substrate, and LgBiT Protein, was added according to manufacturer’s protocol, and cells were incubated for 30 min at ambient temperature with shaking. In some experiments, research-grade purified LgBiT was used in place of the commercial material at a final concentration of 100 nM (Refer to Supporting Information for purification details). Luminescence was then measured using a GloMax Discover System (Promega) with 0.5 s integration time. Live Cell HiBiT Detection. HiBiT-edited cells were plated in white 96-well tissue culture plates at a density of 2 x 104 cells per well in 100 µl growth medium and transduced with 2% (v/v) BacMam CMVLgBiT reagent (Kempbio, Inc., viral titer approximately 2 x 108 PFU/ml) for 24 h. The medium was then replaced with 150 µl CO2-independent medium (GIBCO) containing 20 µM Nano-Glo Live Cell Ex-4377 (Promega), an esterase-labile time-released substrate.31 Cells were incubated for 30 min at 37 °C and then treated with phenanthroline (20 µM, final concentration in 200 µl) in 50 µl of CO2-independent medium. The plates were immediately sealed with transparent adhesive film and placed into a GloMax Discover System set to a temperature of 37 °C. The plate was read every 6 min for 10 h using an integration time of 0.5 s. To measure the effect of reoxygenation on protein abundance, edited cells were plated in 96-well tissue culture plates at a density of 2 x 104 cells per well in 100 µl medium and transduced with 2% (v/v) BacMam CMV-LgBiT reagent for 24 h. The cells were then transferred to a hypoxia chamber for 20 h. Immediately after removing the cells from the chamber, 25 µL of Nano-Glo Live Cell Assay reagent (Promega) was added. Luminescence was then measured every 30 s for 20 min using a GloMax Discover System set to a temperature of 37 oC using an integration time of 0.5 s. NanoBRET Immunoassay for Analysis of Post-Translational Modifications. HeLa HIF1α-HiBiT cells were plated in a white 96-well tissue culture plate (4 x 104 cells per well) and incubated for 24 h at 37 oC. The cells were then treated with 3 µM MG132 and a serial dilution of phenanthroline for 6 h. The medium was replaced with 25 µl assay buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.8, 5 mM EDTA, and 1% NP-40) containing rabbit anti-human hydroxy-HIF1α (Pro564) antibody (1:250 dilution, Cell Signaling Technology) and 100 nM LgBiT. The cells were incubated for 30 min at ambient temperature. After addition of 25 µl assay buffer containing Alexa Fluor 594 labeled anti-rabbit secondary antibody (1:500 dilution, Cell Signaling Technology) and 100 µM furimazine, the samples were incubated for 60 min at ambient temperature. Both total luminescence and BRET signal (acceptor channel 460/60 nm, donor channel 610 nm longpass) were recorded on a GloMax Discover System using 0.5 s integration time. Reagents, Linearity, Blotting, Imaging, and qPCR. See Supporting Information.



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AUTHOR INFORMATION Corresponding Author § E-mail: [email protected]. Notes The authors declare no competing financial interest. *These authors contributed equally to this work.

ACKNOWLEDGEMENTS The authors wish to thank J. Unch for the Nano-Glo Live Cell Ex-4377 substrate and P. Otto for assistance with the purification of LgBiT protein.

ASSOCIATED CONTENT The Supporting Information is available free of charge at http://pubs.acs.org. Figures s1-s11, Tables s1-10, and supporting methods.

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FIGURE LEGENDS Figure 1. Linearity of luminescence generated by HiBiT. (a) Luminescent signal generated either by fulllength recombinant NanoLuc or by Halo-Tag HiBiT incubated with saturating LgBiT. (b) Luminescent signal in live cells and cell lysates. HEK293 cells stably expressing LgBiT were transfected with varying concentrations of an expression plasmid for HaloTag-HiBiT, and luminescence was measured before (live cell) or after (lytic) lysis. For both panels, data represent luminescence after subtraction of background caused by reagent (panel a) or untransfected cells (panel b). Shown are mean values, n = 4, with variability expressed as SD. Figure 2. Quantification of endogenous HIF1α-HiBiT abundance. HIF1α-HiBiT levels in edited HeLa cells were measured in lytic format following 6 h treatment with (a) hypoxia (1% O2) or (b) the following compounds: Phenanthroline (Phen, 20 µM), ML228 (2 µM), DFO (300 µM), IOX2 (100 µM), FG2216 (100 µM), FG4592 (50 µM), DMOG (250 µM), MG132 (10 µM), and MLN4924 (1 µM). (c) HIF1α-HiBiT abundance measured in lytic format following treatment with different concentrations of compound. For panels (a)–(c), shown are mean luminescence values (after subtraction of reagent background) from 4 independent experiments (at least 5 replicates per experiment) with variability expressed as SEM. Dashed line (panel b) denotes luminescence generated by untreated HIF1α-HiBiT-expressing HeLa cells. Figure 3. Hypoxia response of HIF1α target proteins. Levels of endogenous (a) BNIP3-, (b) ANKRD37-, (c) HILPDA-, and (d) KLF10-HiBiT in HeLa cells following 20 h hypoxia treatment. Shown are mean luminescence values (after subtraction of background) from 5 independent experiments (at least 5 replicates per experiment), with variability expressed as SEM. Figure 4. Response of HIF1α target proteins to hypoxia mimetics. Levels of endogenous (a) BNIP3-, (b) ANKRD37-, (c) HILPDA-, and (d) KLF10-HiBiT in HeLa cells following 24 h treatment with the following compounds: Phenanthroline (Phen, 20 µM), ML228 (2 µM), DFO (300 µM), IOX2 (100 µM), FG2216 (100 µM), FG4592 (50 µM), DMOG (250 µM), MG132 (10 µM), and MLN4924 (1 µM). Data represent luminescence acquired in lytic format after background subtraction from 5 independent experiments, with variability expressed as SEM. Dashed line denotes luminescence generated by untreated HiBiTedited cells. Figure 5. Dose response of HIF1α target proteins. Compound-induced accumulation of endogenous (a) BNIP3-, (b) ANKRD37-, (c) HILPDA-, and (d) KLF10-HiBiT in edited HeLa cells treated for 20 h with hypoxia mimetics. Shown are mean luminescence values (after subtraction of reagent background) from 5 independent experiments (at least 5 replicates per experiment), with variability expressed as SEM. Dashed line denotes luminescence generated by untreated HiBiT-edited HeLa cells. Figure 6. Quantification of HiBiT-tagged proteins in primary cells. Levels of endogenous (a) HIF1α-, (b) BNIP3-, and (c) VEGFA-HiBiT in edited HUVEC cells. Cells were treated for 6 h with Phenanthroline (Phen, 20 µM), ML228 (2 µM), DFO (300 µM), MG132 (10 µM), and MLN4924 (1 µM) and then analyzed for luminescence in lytic format. Data are represented as luminescence with background subtracted, n = 4, with variation expressed as SD. Dashed line indicates luminescence generated by edited HUVEC cells. Figure 7. Real-time measurement of induced changes in protein abundance. (a) HeLa cells expressing HiBiT-edited HIF1α and downstream target proteins were transduced with BacMam-LgBiT, and luminescence was monitored following phenanthroline addition. Signal-to-background (S/B) ratios were



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derived using the formula: (RLUphenanthroline-RLUbackground)/(RLUuntreated-RLUbackground), where background is determined from parental HeLa cells. (b) Edited HeLa cells were transduced with BacMam-LgBiT and incubated for 20 h under hypoxia. Luminescence was measured immediately following release from the hypoxia chamber. Shown are mean relative responses, n = 3, with variability represented as SD. Figure 8. Simultaneous quantification of HIF1α hydroxylation at proline 564 and protein abundance. HeLa cells expressing HIF1α-HiBiT were treated for 6 h with phenanthroline in the presence of MG132. HIF1α proline hydroxylation levels are represented as BRET ratio (Acceptor Emission 610 nm/Donor Emission 460 nm; red circles). HIF1α abundance is represented as luminescence (blue circles). Shown are the mean values, n = 3, with variability represented as SD.


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CRISPR-Mediated Tagging of Endogenous Proteins with a

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