EXPRESSION IN MAMMALIAN CELLS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial

No. 62/321,559, filed April 12, 2016, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under grant number

RO1GM078465 awarded by the National Institutes of Health. The government has certain rights in this invention.

FIELD OF THE INVENTION [0003] The present invention relates a system for F-box hormone receptor regulated protein expression in mammalian cells.

BACKGROUND OF THE INVENTION

[0004] Biologists are increasingly adopting holistic approaches, such as systems biology, to understand life's complexity. Nevertheless, reductionism still remains a primary driving force for scientific progress. Elucidating gene function underlies most biological discoveries and is frequently achieved using loss-of-function analyses. Yet, for mammalian cells in general, and even more so for mammalian stem cells, the biologist's toolbox is limited and primarily includes laborious genomic editing (Kim et al., "Genomic Editing Tools to Model Human Diseases with Isogenic Pluripotent Stem Cells," Stem Cells Dev. 23 :2673-2686 (2014)), a limited set of often- nonspecific chemical inhibitors and RNA interference (RNAi). Recently developed tools augment experimental flexibility and accuracy (Auslander et al., "From Gene Switches to Mammalian Designer Cells: Present and Future Prospects," Trends Biotechn. 31 : 155-168 (2013) and Lienert et al., "Synthetic Biology in Mammalian Cells: Next Generation Research Tools and Therapeutics," Nat. Rev. Mol. Cell. Biol. 15:95-107 (2014)), but are still limited in applicability, reversibility, titratability, rapidity, and multiplicity (Table 1). Table 1. Selected Methods to Regulate Gene Activity in Mammalian Cells.

* Nishimura et al., "An Auxin-Based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009).

i. RNAi can affect hundreds of genes. To overcome this, different RNAi sequences targeting the same gene can be used to substantiate causality between silencing and observed phenotypes (Cullen et al., "Enhancing and Confirming the Specificity of RNAi Experiments," Nat. Methods 3:677-681 (2006)).

ii. Effective gene silencing is usually obtained within 2-3 days (Brummelkamp et al., "A System for Stable Expression of Short Interfering RNAs in Mammalian Cells," Science 296:550-553 (2002) and van de Wetering et al., "Specific Inhibition of Gene Expression Using a Stably Integrated, Inducible Small Interfering-RNA vector," EMBO Reports 4:609-615 (2003)). With inducible systems, silencing can be obtained within 1-2 days and reversal of the effect usually takes longer.

iii. Tet-regulated systems require rtTA or tTA, which usually necessitates the delivery of a second plasmid encoding one of these proteins, or a specialized cell type that stably expresses it. iv. If an RNAi-dependent phenotype is reversed by the CDS of the silenced gene, it is unlikely that the phenotype stemmed from an RNAi off-target effect (Cullen et al., "Enhancing and Confirming the Specificity of RNAi Experiments," Nat. Methods 3:677-681 (2006)).

v. Tet-induced expression is usually hard to accurately titrate.

vi. A rescue (or genetic complementation) system enables the replacement of an endogenous gene with an exogenous version that can be regulated externally. Represents a type of a molecular switch.

vii. pAID allows expression of auxin-degradable proteins. However, it does not contain a component, such as an shRNA, that inactivates an endogenous gene and allows its replacement by the auxin-degradable protein.

viii. The AID degron used in pAID is the full-length thaliana IAA17 transcription factor. As a degron, it suffers from several disadvantages, including its large size (228-amino acids), its ability to confer nuclear localization to the fused POI (data 14 not shown) and, possibly, a tendency to be spontaneously cleaved-off from the POI. Notably, the observation of spontaneous cleavage of GFP-AID ²²⁸ in pRAIDRS-infected 293T cells (FIGs 2 A-2F) was not reported by other groups, who successfully used GFP-AID ²²⁸ .

ix. Non-viral plasmids are hard to deliver into some types of mammalian cells, including hard-to-transfect cells (such as fibroblasts, ESCs, and many primary cell types) and slow-proliferating cells. Additionally, genomic integration of nonviral plasmids is an extremely rare event in many cell types.

x. The CMV promoter undergoes silencing in certain mammalian stem cells, such as human ESCs

Xia et al, "Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in a Promoter- Dependent Manner," Stem Cells and Development 16: 167-176 (2007) and Norrman et al., "Quantitative Comparison of Constitutive Promoters in Human ES Cells," PLoS ONE 5x12413 (2010).

xi. Auxin-dependent degradation was utilized successfully in several studies of mammalian cells (Han et al.,, "Catalytic

Assembly of the Mitotic Checkpoint Inhibitor BubRl-Cdc20 by a Mad2 -Induced Functional Switch in Cdc20," Mol. Cell 51 :92-104 (2013); Holland et al, "Inducible, Reversible System for the Rapid and Complete Degradation of Proteins in Mammalian Cells," Proc. Natl. Acad. Sci. USA 109:E3350-3357 (2012); Lambrus et al., "p53 Protects Against Genome Instability Following Centriole Duplication Failure," J. Cell Biol. 210:63-77 (2015); and Rodriguez-Bravo et al, "Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Madl- Dependent Anaphase Inhibitor," Cell 156: 1017- 1031 (2014)), albeit not in stem cells. Nevertheless, to engineer auxin-degradable proteins in mammalian cells the authors had to use several consecutive and rather-laborious steps. For example, Holland et al, "Inducible, Reversible System for the Rapid and Complete Degradation of Proteins in Mammalian Cells," Proc. Natl. Acad. Sci. USA 109:E3350-3357 (2012) as well as Han et al., "Catalytic Assembly of the Mitotic Checkpoint Inhibitor BubRl-Cdc20 by a Mad2 -Induced Functional Switch in Cdc20," Mol. Cell 51 :92-104 (2013) first generated cell lines that overexpress the TIR1 receptor, then overexpressed an AID-fused POI, and finally transiently knocked-down the gene encoding for the endogenous POI.

Similarly, Lambrus et al, "p53 Protects Against Genome Instability Following Centriole Duplication Failure," J. Cell Biol. 210:63-77 (2015) sequentially targeted the AID degron to the two endogenous alleles of the POI, and then overexpressed TIR1.

xii. The system is limited to exogenously-expressed proteins, unless genome-editing is used to fuse the destabilizing FKBP domain to both alleles of an endogenous gene, or a form of rescue system is established by inactivating the endogenous gene and replacing it with an FKBP -fused version.

xiii. pRAIDRS and pJAZ harbor relatively short degrons (47-amino acids and 33-amino acids, respectively). Shorter degrons reduce the likelihood of steric interference with the POPs function, and supposedly, have less non-degron functions (such as interactions with additional proteins or DNA or effect on protein localization). Additionally, the shorter AID degron seem to suffer less from spontaneous cleavage from the POI compared with the full-length AID ²²⁸ , at least in our experimental settings (FIGs 2C-2D).

xiv. As opposed to the RNAi+Tet-Inducible CDS Rescue System, pRAIDRS and pJAZ contain all the necessary genetic

elements to silence an endogenous gene and replace it by a hormone-degradable POI. They can be used in nonspecialized cells without additional components.

xv. In pRAIDRS and pJAZ the selectable markers (either PuroR or BSD genes) are cloned in-frame with the hormone receptor and degron-POI. Following translation and cleavage at the P2A peptides, the selectable marker is released and can render cells resistant to its corresponding drug. The expression of all components from a single promoter and as a single pre- processed protein reduces the likelihood that in drug-resistant cells will silencing or mutation the hormone receptor or degron-POI will occur.

xvi. Both pRAIDRS and pJAZ show increased efficiency with nuclear POIs compared to cytoplasmic POIs.

xvii. Coronatine-dependent degradation in mouse embryonic stem cells is ineffective (usually 50-80%, compared with >90% in other mouse cell types and all tested human cell types).

Thus, simple tools for rapid and multiple gene perturbation will facilitate the elucidation of gene functions and molecular networks.

[0005] Manipulation of protein levels represents a relatively new loss-of-function approach. To this end, harnessing the plant hormone-induced degradation pathways is particularly attractive due to its efficiency and specificity. The plant hormones auxin (indole-3- acetic acid, "IAA") and jasmonate-isoleucine (jasmonic acid-Ile, "JA-De") bind the intracellular F-Box proteins transport inhibitor response 1 ("TIR1") and coronatine insensitive 1 ("COH"), respectively, and promote their association with target proteins containing specific degron motifs. TIR1 and COI1, via their F-box domains, assemble into the SCF (SKP1, CUL1 and F- box) E3 ubiquitin-ligase complex, which together with an E2 ubiquitin-conjugating enzyme, catalyses the polyubiquitination and subsequent proteasomal degradation of degron-containing proteins (Tan et al., "Mechanism of Auxin Perception by the TIR1 Ubiquitin Ligase," Nature 446:640-645 (2007); Sheard et al., "Jasmonate Perception by Inositol-phosphate-potentiated COI1-JAZ Co-Receptor," Nature 468:400-405 (2010); Dharmasiri et al., "The F-Box Protein TIR1 is an Auxin Receptor," Nature 435:441-445 (2005); Kepinski et al., "The Arabidopsis F- Box Protein TIR1 is an Auxin Receptor," Nature 435:446-451 (2005); Thines et al., "JAZ Repressor Proteins are Targets of the SCF(COIl) Complex During Jasmonate Signalling," Nature 448:661-665 (2007); and Chini et al., "The JAZ Family of Repressors is the Missing Link in Jasmonate Signalling," Nature 448:666-671 (2007)). Auxin-bound TIRl targets proteins containing auxin-induced degradation ("AID") degrons, while JA-Ile-bound COI1 targets proteins containing JAZ degrons (FIG 1A). Nishimura et al., "An Auxin-based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009) developed a system enabling conditional protein regulation by adapting the auxin-induced degradation pathway to non-plant cells. They reported that ectopic TIRl can mediate auxin- dependent degradation of AID-fused proteins and demonstrated the system's feasibility with a simple plasmid (pAID) harboring a cytomegalovirus promoter-driven polycistronic mRNA encoding TIRl and a plant protein carrying the AID degron. Fusing a protein-of-interest ("POI") to the degron enabled the degradation of the degron-fused POI following auxin treatment (Nishimura et al., "An Auxin-based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009)). Despite its simplicity, pAID has major limitations in terms of applicability to mammalian cells. These include a viral promoter prone to silencing in embryonic stem cells ("ESCs") (Norrman et al., "Quantitative Comparison of Constitutive Promoters in Human ES Cells," PLoS ONE 5:el2413 (2010) and Xia et al., "Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in a Promoter-dependent Manner," Stem Cells Dev. 16: 167-176 (2007)), a lack of a designated selectable marker, the inability to suppress endogenous genes and a large degron (228 amino acids) liable to interfere with the POI's function. For these and other reasons (Table 1), this technology has been primarily applied to yeast, where endogenous genes are easily disrupted and pAID-carrying clones are readily isolated. Of note, although auxin-dependent degradation was previously used to study mammalian cells, its implementation required multiple consecutive genetic manipulations and was mainly confined to cancer cell lines (Han et al., "Catalytic Assembly of the Mitotic Checkpoint Inhibitor BubRl-Cdc20 by a Mad2-Induced Functional Switch in Cdc20," Mol. Cell 51 :92-104 (2013); Holland et al., "Inducible, Reversible System for the Rapid and Complete Degradation of Proteins in Mammalian Cells," Proc. Natl. Acad. Sci. USA 109:E3350-3357 (2012); Rodriguez-Bravo et al., "Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad 1 -dependent Anaphase Inhibitor," Cell 156: 1017-1031 (2014); and Lambrus et al., "p53 Protects Against Genome Instability Following Centriole Duplication Failure," J. Cell Biol. 210:63-77 (2015)). In recent times, auxin-dependent degradation was also harnessed in vivo to study Caenorhabditis elegans (Zhang et al., "The Auxin-Inducible Degradation (AID) System Enables Versatile Conditional Protein Depletion in C. elegans;' Development 142:4374-4384 (2015)).

[0006] Mammalian ESCs have gained much interest as a model for developmental biology and a therapeutic avenue. ESCs are unique in their unlimited self-renewal and plunpotency, a state maintained by a transcription factor network revolving around SOX2, OCT4 (POU5F1) and NANOG (Macarthur et al., "Systems Biology of Stem Cell Fate and Cellular Reprogramming," Nat Rev Mol Cell Biol 10:672-681 (2009)). Combining loss-of-function and genetic complementation (rescue) strategies, the ESC self-renewal network was broadened and characterized (Macarthur et al., "Systems Biology of Stem Cell Fate and Cellular

Reprogramming," Nat Rev Mol Cell Biol 10:672-681 (2009); Gingold et al., "A Genome-wide RNAi Screen Identifies Opposing Functions of Snail and Snai2 on the Nanog Dependency in Reprogramming," Mol Cell 56: 140-152 (2014); Lee et al., "Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling," Cell Stem Cell 11 : 179-194 (2012); Ang et al., "Wdr5 Mediates Self-Renewal and Reprogramming Via the Embryonic Stem Cell Core Transcriptional Network," Cell 145: 183-197 (2011); and Ivanova et al., "Dissecting Self- Renewal in Stem Cells with RNA Interference," Nature 442:533-538 (2006)). There is a need for an improved experimental system that upgrades the stem cell biologist's toolbox and facilitates faster, tighter and combinatorial dissection of gene and protein function.

[0007] The present invention is directed to overcoming the above-noted deficiencies in the art.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a system for F-box hormone receptor regulated protein expression. The system includes a silencing nucleic acid molecule comprising a first promoter and an shRNA operably linked to the first promoter, where the shRNA silences expression of a target protein. The system also includes an expression nucleic acid molecule comprising a second promoter, an F-box hormone receptor operably linked to the second promoter, and a nucleic acid molecule encoding a fusion protein comprising a degron fused to the target protein, where the nucleic acid molecule encoding the fusion protein is operably linked to the second promoter.

[0009] The present invention also relates to a vector comprising the system of the present application, where the silencing nucleic acid molecule is coupled directly or indirectly to the expression nucleic acid molecule.

[0010] The present invention further describes a method for F-box hormone receptor regulated protein degradation in a mammalian host cell. This method involves providing a mammalian host cell; introducing the system of the present application into the mammalian host cell to produce a transgenic mammalian host cell; culturing said transgenic mammalian host cell under conditions that result in (i) silencing expression of the target protein and (ii) expression of the fusion protein; and contacting said transgenic mammalian host cell with a molecule that binds the F-box hormone receptor so that said fusion protein undergoes degradation.

[0011] The present invention also describes a method for F-box hormone receptor regulated target protein degradation in a mammalian host cell. This method involves providing a mammalian host cell and infecting, into the mammalian host cell, a first lentiviral vector comprising (i) a first silencing nucleic acid molecule comprising a primary first promoter and a first shRNA operably linked to the primary first promoter, where the first shRNA silences expression of a first target protein and (ii) a first expression nucleic acid molecule comprising a primary second promoter, a Transport Inhibitor Response 1 ("TIRl") receptor operably linked to the primary second promoter, and a nucleic acid molecule encoding a first fusion protein comprising an auxin-induced degradation ("AID") degron fused to a first target protein, where the nucleic acid molecule encoding the first fusion protein is operably linked to the primary second promoter. The method further involves infecting, into the mammalian host cell, a second lentiviral vector comprising (i) a second silencing nucleic acid molecule comprising a secondary first promoter and a second shRNA operably linked to the secondary first promoter, where the second shRNA silences expression of a second target protein and (ii) a second expression nucleic acid molecule comprising a secondary second promoter, a Coronatine Insensitive 1 ("COH") receptor operably linked to the secondary second promoter, and a nucleic acid molecule encoding a second fusion protein comprising a JAZ degron fused to a second target protein, where the nucleic acid molecule encoding the second fusion protein is operably linked to the secondary second promoter. The method further involves culturing the infected mammalian host cell under conditions that result in (i) silencing expression of the first and second target proteins and (ii) expression of the first and second fusion proteins; contacting the infected mammalian host cell with a molecule that binds the TIR1 receptor so that said first fusion protein undergoes degradation; and contacting the infected mammalian host cell with a molecule that binds the COI1 receptor so that said second fusion protein undergoes degradation.

[0012] The examples of the present application describe a mammalian dual-protein rescue system that harnesses the auxin and JA-Ile pathways, and may be specifically tailored to ESCs. For each hormone, a lentiviral vector harboring a short hairpin RNA ("shRNA"), a hormone receptor, a short degron and a selectable marker was engineered. Using a two-step cloning protocol, each vector is easily modified to contain the desired shRNA and degron-fused POI, which enables silencing of a gene-of-interest and its replacement by a POI whose degradation is induced by the appropriate hormone. The combination of these two vectors offers simultaneous control over two proteins in the same cell. By applying this system to study key ESC decision-making proteins, such as NANOG, CHKl, p53 and NOTCH1, the system's potential to facilitate experimental designs that were previously unfeasible or overcomplicated are demonstrated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGs 1 A-1B illustrate a mechanisms of plant hormone-induced degradation and the structure of plant hormone-induced degradation vectors. FIG 1 A is a schematic illustration of plant hormone-induced protein degradation pathways. The plant SCF E3 ubiquitin ligase complex comprises SKP1, CUL1, and an F-box hormone receptor. On binding its cognate hormone, the receptor recruits the SCF complex to a target protein containing a degron motif. A recruited E2 ubiquitin-conjugating enzyme ubiquitinates the target, leading to its rapid proteasomal degradation. Hormone ("H"); ubiquitin ("Ub"). FIG IB, upper panel is a schematic representation of the pRAIDRS and pJAZ vector structure. FIG IB, bottom, shows pre- and post-P2A-mediated processing of the translated components. The two-headed arrow indicates that the degron can be fused to either terminus of the POI. Ampicillin resistance β-lactamase ("AmpR"); long-terminal repeat ("LTR"); multiple cloning site ("MCS"); protein-of-interest ("POI"); Psi packaging signal ("Psi"); Rev response element ("RRE"). See also FIGs 2A-2F.

[0014] FIGs 2A-2F show the optimization and characterization of pRAIDRS. FIG 2A is a graph showing Green Fluorescent Protein ("GFP") expression of cells infected with lentiviral vectors using pRAIDRS harboring either a PGK-lor an EFla promoter and GFP-AID ⁴⁷ into CCE and Rl mESCs or H9 hESCs. Cells were selected, treated with 0.01% EtOH ("Con") or 50 μΜ auxin ("IAA") and analyzed for GFP fluorescence by flow cytometry. For each cell type, values were normalized to the "pPGK-1, Con" sample. Data represent averages and standard deviation of 2 biological replicates. FIG 2B is a sequence alignment showing a conserved region among three A. thaliana auxin-degradable proteins ( ΛΑΑ14, NP 193191 (SEQ ID NO: 1); ΑΛΑΑΊ, NP_188945( SEQ ID NO: 2); Atl Ml, NP_171921 (SEQ ID NO: 3)) (Villalobos et al, "A Combinatoria TIRl/AFB-Aux/IAA Co-Receptor System for Differential Sensing of Auxin," Nat. Chem. Biol. 8:477-485 (2012), which is hereby incorporated by reference in its entirety) and three versions of _^ 4tIAA17 that were tested as degrons in pRAIDRS. Conserved residues are highlighted in red. Superscript numbers in degron names indicate length in amino acids. FIG 2C is a graph showing auxin-induced degradation mediated by different AID degrons. HEK- 293T cells were infected with pRAIDRS vectors harboring a GFP fused to degrons of different sizes. Cells were treated with increasing concentrations of IAA and analyzed 24 hours later (left panel) or with 500 μΜ IAA for the indicated time periods (right panel). GFP fluorescence was analyzed by flow cytometry. Data series are color-coded as in FIG 2B. Experiment was conducted twice and representative results are displayed. FIG 3D shows a western blot analysis of GFP in pRAIDRS-infected HEK-293T cells. Untreated cells are expressing GFP fused to either the full-length ^tIAA17 ("AID ²²⁸ ") or to AID ⁴⁷ . The bands corresponding to GFP-AID ²²⁸ and GFP-AID ⁴⁷ are marked with a single asterisk at an expected molecular weight of 58 and 36 kDa, respectively. An additional band (two asterisks) corresponds to cleaved (unfused) GFP protein with the expected molecular weight of 27 kDa. Experiment was conducted 2 times and representative results are displayed. Note the complete absence of cleaved GFP in the right lane, indicating that this spontaneous cleavage occurs only with AID ²²⁸ . This phenomenon can explain the biphasic kinetics of GFP-AID ²²⁸ degradation (FIG 2B) whereas the fused GFP is rapidly degraded, while the unfused is insensitive to auxin. FIG 2E is a graph showing the effect of nuclear localization on auxin- induced degradation of GFP-AID ⁴⁷ . HEK-293T cells were infected with pRAIDRS containing either GFP-AID ⁴⁷ or NLS-GFP-AID ⁴⁷ . Cells were treated with increasing concentrations of IAA and analyzed after 24 hours (left panel), or with 50 μΜ IAA for the indicated time periods (right panel). GFP fluorescence was analyzed by flow cytometry. The experiment was conducted 3 times, and representative results are displayed. FIG 2F shows GFP and DAPI microscopic images of HEK-293T cells infected with pRAIDRS containing either GFP-AID ⁴⁷ or NLS-GFP-AID ⁴⁷ . [0015] FIGs 3 A-3D illustrate an auxin-degradable NANOG rescue system in mESCs.

CCE mESCs were infected with pRAIDRS AID ⁴⁷ -NANOG (A-NANOG) or GFP-AID ⁴⁷ ("GFP-A") and selected clones with ESC morphology were analyzed for the effect of auxin ("IAA") treatment. FIG 3 A is a western blot analysis depicting endogenous NANOG (*) and A-NANOG (**) in parental mESCs, and in the indicated clones. β-Actin serves as a loading control. Experiment was repeated three times and a representative blot is presented. FIG 3B are images of mESC clones plated at low density, grown in the presence of ethanol ("Con") or auxin ("IAA") for 3-4 days and assayed for AP activity. GFP-A mESCs reached the desired confluency a day earlier and therefore the images were taken on different days. FIG 3C are bright-field images showing representative morphology of A-NANOG mESCs following 3 days of ethanol ("Con") or auxin ("IAA") treatment (upper part) and merged bright-field and GFP fluorescence images of ethanol or auxin-treated GFP-A mESCs (lower part). Scale bars, 100 μπι. FIG 3D shows a heatmap of mESC clones treated with auxin for the indicated number of days. All cells were subjected to the same concentration of ethanol for the duration of experiment. Quantitative real-time PCR analysis was performed for selected self-renewal and differentiation markers, and normalized expression levels are represented as a heatmap. For FIGs 3B-3D, the differentiation experiment was repeated two times and representative results are displayed. See also FIGs 4A-4F and FIG 15.

[0016] FIGs 4A-4F illustrate an auxin-degradable NANOG rescue system in mESCs. In FIG 4A, A-NANOG #2 mESCs were treated with 50 μΜ auxin ("IAA") for the indicated time periods. Western blot analysis demonstrates complete depletion of ANANOG following 30 minutes of auxin treatment. Endogenous NANOG and exogenous A-NANOG are marked by * and **, respectively. The experiment was conducted 3, times and representative results are displayed. FIG 4B shows selected mRNA expression patterns from the heatmap in FIG 3D displayed as bar charts. Error bars represent standard deviation of three technical replicates. FIGs 4C-4D illustrate shRNA-mediated knockdown of NANOG in CCE mESCs. Cells were infected with pLKO. l-Puro-IRESmCheriy harboring either a Luciferase shRNA ("sh-Con") or a Nanog shRNA ("sh-Nanog"). Two days post infection cells were selected with 1 μg/ml

Puromycin for the indicated number of days. On the third day of selection, cells were collected for the first QRT-PCR analysis time point and the rest of the cells were replated for the following time points. FIG 4C is a western blot analysis of cells collected on the fifth day of selection. FIG 4D shows QRT-PCR data presented as a heatmap. Experiment was conducted twice and representative results are displayed. In FIG 4E, mESCs were infected with either pRAIDRS GFP-AID ⁴⁷ ("GFP-A"), pRAIDRS AID ⁴⁷ -NANOG ("A-NANOG") or pRAIDRS OsJAZ ³³ - NANOG ("J-NANOG"), which harbors a Nanog shRNA and a Nanog coding sequence fused to the OsJAZ ³³ degron. In the latter cells, endogenous Nanog is replaced by an exogenous Nanog that does not contain an AID degron and, therefore, should be auxin resistant. A western blot analysis depicts endogenous NANOG (*) and exogenous degron-fused NANOG (**) in a pool of GFP-A mESCs and clones of ANANOG and J-NANOG mESCs. Experiment was conducted once. FIG 4F are images of GFP-A (pool) and clones of A-NANOG and JNANOG mESCs were plated at low density, grown in the presence of EtOH ("Con") or auxin ("IAA") for 4 days and assayed for alkaline phosphatase activity. Low magnification pictures were taken using a bright- field microscope. Note that only A-NANOG mESCs demonstrated reduction in AP -positive colony number upon auxin treatment.

[0017] FIGs 5A-5F show that rapid CHK1 depletion sensitizes mESCs to DNA damage.

FIG 5A is a western blot analysis depicting endogenous CHK1 (*) and AID ⁴⁷ -CHK1 (A-CHKl, **) in four selected clones and in parental non-infected mESCs. β-Actin serves as a loading control. Experiment was repeated three times for clones #1 and #2, and a representative blot is presented. In FIG 5B, A-CHKl #2 mESCs were treated with ethanol ("Con") or auxin ("IAA") for 1 day. Cells were then treated with the indicated concentrations of aphidicolin ("Aph"). Equal concentrations of DMSO were applied to all conditions. The next day, cells were stained with crystal violet and plates were scanned. The experiment was repeated three times, and a representative result is displayed. In FIG 5C, mESC clones were pretreated with 1 mM aphidicolin for 1 day and were then treated with auxin for the indicated time periods. Left panel: mitotic index was calculated as the percentage of H3 ^pS10 -positive cells with 4N DNA content, measured by flow cytometry. Right panel: dot plots for A-CHKl #2 mESCs treated with auxin for 0 or 45 minutes. Mitotic cells are gated. FIG 5D show bright-field microscope images of cells treated as in FIG 5C. Images show synchronous cell rounding, a feature of late mitotic cells, 2 hours following auxin treatment in aphidicolin-treated A-CHKl #2 cells. FIG 5E is a western blot analysis of cells treated as in FIG 5C. Tyrl5 phosphorylation of CDK1

("CDKl ^pY15 ") was detected using a phospho-specific antibody. β-Actin serves as a loading control. FIG 5F shows quantitative real-time PCR analysis of Fas mRNA in cells treated as described in FIG 5C. Error bars represent standard deviation of three technical replicates. For FIGs 5C-5F, kinetic experiments was repeated three times and representative results are displayed. See also FIGs 6A-6E, 7A-7G, 8A-8C, and 15.

[0018] FIGs 6A-6E show CHK1 depletion under normal growth conditions in mESCs.

FIG 6A show images of cells assayed for alkaline phosphatase activity. CCE mESCs were infected with pRAIDRS GFP-A or A-CHKl and selected in the presence of EtOH ("Con") or auxin ("IAA"). After colonies emerged, cells were assayed for alkaline phosphatase activity. Left, low magnification scans. Right, bright-field microscope images. Scale bars, 100 μπι. The experiment was conducted 3 times, and representative results are displayed. FIG 6B are graphs showing populations doubling, following selection, of the indicated clones cultured in the presence of 0.01% EtOH (solid lines) or 50 μΜ IAA (dashed lines). Cells were counted and replated every 3-4 days and population doublings were calculated as Log ₂ (cell output/cell input). Experiment was conducted once for these clones. In FIG 6C, CCE A-CHKl clone #2 cells were subjected to a competition assay (Lee et al., "Combining Competition Assays with Genetic Complementation Strategies to Dissect Mouse Embryonic Stem Cell Self-Renewal and

Pluripotency," Nat. Protoc. 7:729-748 (2012), which is hereby incorporated by reference in its entieritey.) Cells were labeled with mCherry fluorescence protein and co-cultured with control GFP-A CCE cells in the presence of 0.01% EtOH or 50 μΜ IAA. Cells were collected every 2-3 days and assayed for GFP and mCherry fluorescence by flow cytometry. The percentage of A- CHK1 cells was calculated for each time point and the values for the auxin-treated cells were normalized to those of EtOH-treated cells. An average growth rate decrease of 8% per day was calculated for auxin-treated A-CHKl cells compared to the EtOH-treated controls. For comparison, a similar assay performed by Ivanova et al. reported drastic reduction in cell proportion following knock-down of genes involved in mESC self-renewal (Ivanova et al., "Dissecting Self-Renewal in Stem Cells with RNA Interference," Nature 442: 533-538 (2006), which is hereby incorporated by reference in its entirety). The experiment was conducted twice, and representative results are displayed. In FIG 6D, the indicated clones were treated with 0.01%) EtOH (orange histograms) or 50 μΜ IAA (blue histograms) for 2 days and analyzed for cell surface pluripotency marker SSEA-1 expression by flow cytometry. Red histogram, isotype control. Numbers indicate percentages of SSEA-1 positive cells and are color-coded similarly to the histograms. Experiment was conducted once. In FIG 6E, the indicated clones were treated with 0.01%) EtOH or 50 μΜ auxin ("IAA") for 3 days and analyzed for the expression of self- renewal and differentiation markers by QRT-PCR. Bars represent Log ₂ (fold

change[IAA/EtOH]). Error bars represent standard deviation of 3 technical replicates. Auxin- induced differentiation experiment was performed 3 times and representative results are displayed. On the right, data from retinoic acid ("RA", 5 μΜ, 3 days) and EtOH ("Con", 0.05%, 3 days) treated mESCs serve as a positive control for mESC differentiation transcriptional alterations.

[0019] FIGs 7A-7G show that CHK1 protects mESCs from aphidicolin-induced death and differentiation. CCE mESCs were infected with pRAIDRS A-CHKl or GFP-A. Selected clones were analyzed for their CHK1 -dependent response to aphidicolin treatment. FIG 7 A are images of cell stained with crystal violet. The indicated clones were pre-treated with 0.01% EtOH or 50 μΜ auxin ("IAA") for one day. Cells were then treated with the indicated concentrations of aphidicolin ("Aph"). Equal concentrations of DMSO were applied in all conditions. The next day, cells were stained with crystal violet and plates were scanned. FIG 7B shows cell stained with crystal violet. The indicated clones were pre-treated with 0.01% EtOH or 50 μΜ auxin ("IAA") for one day. Cells were then treated with 1 μΜ aphidicolin ("Aph") or 0.01%) DMSO for 12 hours, trypsinized, resuspended in fresh media and 5% of the total volume was replated. Replated cells were grown in the absence of aphidicolin for two days, stained with crystal violet and microscope images were acquired. Scale bars, 200 μιη. Experiment was conducted 2 times and representative results are displayed. In FIG 7C, the indicated mESC clones were pre-treated with EtOH or auxin (IAA) for one day. Cells were then treated with 1 μΜ 5 aphidicolin ("Aph") or 0.001% DMSO for 12 hours and analyzed for DNA content using propidium iodide ("PI"). The percentage of cells with less than 2N DNA content (% SubGl cells) was plotted. The experiment was repeated 2 times, and representative results are displayed. FIGs 7D-7E show that CHK1 depletion in aphidicolin-treated cells leads to induction of p53 transcriptional targets and differentiation markers. The indicated mESC clones were pre- treated with 0.01%) EtOH or 50 μΜ IAA for one day and were then treated with 1 μΜ

aphidicolin ("Aph") or 0.01% DMSO for 12 or 24 hours. QRT-PCR analysis was performed for selected markers, including p53 target genes (Fas, Mdm2, p21, Noxa), endodermal

differentiation markers (Gata4, Sox 17), mesodermal differentiation markers (T, Mixll), ectodermal differentiation marker (Nestin) and trophectodermal differentiation markers (Cdx2, Handl). FIG 7D shows a heat map of normalized average expression levels. FIG 7E are bar charts showing the expression levels of selected genes. Experiment was repeated 3 times and representative results are displayed. FIG 7F is a graph of the apoptotic index of the cells. The indicated clones were pre-treated with 1 μΜ aphidicolin for one day and were then treated with EtOH ("Con") or auxin ("IAA") for the indicated time periods. Apoptotic index was calculated as the percentage of Annexin V-positive, 7-AAD-negative cells. Experiment was repeated twice and representative results are displayed. In FIG 7G, cells were treated as described above.

Bright-field microscope images showing synchronous cell rounding, a feature of late mitotic cells, two hours following auxin treatment in aphidicolin-treated ACHK1#2 cells, but not in GFP-A #5 cells. Experiment was performed more than 3 times and representative results are displayed. [0020] FIGs 8A-8C show that pRAIDRS Enables Titratable Regulation of Protein Level.

FIG 8A shows a western blot image and chart of the protein levels of HEK-293T cells infected with pRAIDRS GFP-AID ⁴⁷ and treated with the indicated concentrations of IAA for one day. β- Actin was used as a loading control. Protein levels were quantified and normalized levels were presented in the chart shown below the western blot image. Experiment was performed 3 times and representative results are displayed. FIG 8B shows a western blot image and chart of the protein levels of CCE mESCs clones GFP-A #5 and A-CHK1 #4 treated with the indicated concentrations of IAA for one day. a-Tubulin was used as a loading control. Protein levels were quantified and relative levels of total CHK1 (calculated as the level of endogenous CHK1 (*) plus the level of A-CHK1(**) divided by the level of a-Tubulin and normalized so that endogenous CHK1 in GFP #5 was set to 1) are plotted (bottom). In FIG 8C, cells were treated with increasing concentrations of auxin (as in FIG 8B). The next day, media was supplemented with 1 μΜ aphidicolin ("Aph") or 0.01% DMSO ("Con") for 24 hours. Cells were stained with crystal violet, washed, and the remaining crystal violet was extracted with acetic acid and quantified using a spectrophotometer at 590 nm. Relative cell number was calculated as the ratios of Aph/Con-treated samples. Error bars represent standard deviation of 3 technical replicates. Statistical significance was calculated using a non-paired t-test for each IAA concentration compared with concentration 0 μΜ and statistically significant p-values (<0.05) are proved next to their corresponding bars. Experiment was performed twice and representative results are displayed.

[0021] FIGs 9A-9D show that pRAIDRS enables reversible regulation of protein level.

NCI-H1299 p53-null lung adenocarcinoma cells were infected with pRAIDRS p53-AID ⁴⁷ ("p53- A") or mOrange-AID ⁴⁷ ("mOrange-A") as a control. Cells were maintained with 200 mM auxin, to constantly induce p53 degradation. FIG 9A is a western blot analysis of p53 and p21 of cells washed three times and incubated with fresh media in the absence of auxin for the indicated time periods. β-Actin serves as a loading control. FIG 9B is a quantification of protein levels presented in FIG 9A. Values were normalized such that the level of each protein at the 24-hour time point was set to 100%. FIG 9C shows quantitative real-time PCR analysis of the p53 target genes p21 ("CDKN1 A") and MDM2. Cells were treated as described above prior to analysis. Error bars represent standard deviation of three technical replicates. The experiment was repeated three times, and representative results are displayed. FIG 9D shows the populations doublings of cells grown in the presence of ethanol ("Con") or auxin ("IAA") and counted every day for 4 days. Media was replaced daily. Population doublings ("PDLs") were calculated as Log ₂ (cell output/cell input). Error bars represent standard deviation of three technical replicates. The experiment was repeated twice, and representative results are displayed. See also FIG 15.

[0022] FIGs 10A-10M shows the optimization and characterization of pJAZ. FIG 10A is a sequence alignment of O. sativa coronatine-degradable proteins (Tan et al., "Mechanism of Auxin Perception by the THU Ubiquitin Ligase," Nature 446:640-645 (2007), which is hereby incorporated by reference in its entirety) OsJAZl (NP_001064513.1; SEQ ID NO: 4), OsJAZ2 (NP_001049167.1; SEQ ID NO: 5), OsJAZ7 (NP_001063273.1; SEQ ID NO: 6), OsJAZ3 (NP_001049166.1; SEQ ID NO: 7), and thaliana AtJAZl (NP_564075; SEQ ID NO: 8). Degron sequences used in this study are highlighted and degron names indicated on the right. FIG 10B is a sequence alignment of the F-box domains used in the present application: hsSKP2 (NP_005974; SEQ ID NO: 9) amino acids 95-132, AtCOll (NP_565919; SEQ ID NO: 10) amino acids 1-51 and OsTIRl (NP OO 1052659.1; SEQ ID NO: 11) amino acids 1-39. FIGIOC is a schematic representation of the hormone receptor structure of different pJAZ versions. Each receptor is composed of combinations of: human SKP2 FBox domain ("i¾SKP2 ^F"box "), either full-length human SKP1 ("HsSKPl") or HsSKPl lacking its F-box binding region (amino acids 1-129, "HsSKPl^ ^"129 "), rice THU F-box domain ("OsTIRl ^F"box ") and thaliana COI1

Leucine-Rich Repeats (" _^ 4tCOIl ^LRR "). FIG10D is a graph showing the percent degradation in HEK-293T cells infected with the pJAZ versions depicted in FIG IOC. All pJAZ vectors harbored GFP-^tJAZ ²³ . Cells were treated with 0.1% DMSO or 50 μΜ coronatine for 24 hours. GFP level was analyzed by flow cytometry and % degradation was calculated as described in the Methods. The experiment was conducted twice, and error bars represent standard deviation of biological replicates. FIG 10E shows a western blot analysis of HEK-293T cells infected with pJAZ 2 (harboring OsTIRl ^F"box -,4tCOIl ^LRR and GFP-^tJAZ23), pJAZ 2 ^HA (harboring HA- O^TIRl ^F"box -^tCOIl ^LRR and GFP-^tJAZ ²³ ) or pRAIDRS 7 ^HA (harboring HA-OsTIRl and GFP- AID ⁴⁷ ). β-Actin serves as loading control. The experiment was conducted twice, and

representative results are displayed. See FIG 15 for un-cropped blots. FIG 10F show GFP fluorescence histograms derived from flow cytometric analysis of HEK-293T cells infected with the indicated pJAZ versions, selected and treated with either 0.1% DMSO ("Con") or 50 μΜ coronatine ("Cor"). Parental HEK-293T cells were analyzed as a control for background autofluorescence. An arrow points to a population of GFP ^low cells in control -treated cells infected with pJAZ 6-Os23. The top three and bottom two panels derive from different experiments. FIGIOG are GFP and DAPI microscopic images of HEK-293T cells infected with pJAZ 6 harboring GFP fused to the indicated degrons. FIG 10H show the relative median fluorescence of HEK-293T cells infected with pJAZ 7 harboring either GFP-OsJAZ ³³ or NLS- GFP-OsJAZ ³³ . Cells were selected and treated with increasing concentrations of coronatine ("Cor") and analyzed after 24 hours (left panel), or with 50 μΜ coronatine for the indicated time periods (right panel). GFP fluorescence was analyzed by flow cytometry. The experiment was conducted twice, and representative results are displayed. FIG 101 shows that NLS-GFP- OsJAZ33 degradation is dependent on the presence of coronatine and coronatine receptor. HEK- 293T cells were infected in duplicates with pJAZ (version 7) containing an NLS-GFP-OsJAZ ³³ or with the same vector lacking coronatine receptor ("CorR"). To control for the structure and size of the vector, CorR (OsTIRl ^F"box -OsCOIlB ^LRR ) was replaced with an auxin receptor (OsTIRl). Cells were selected and treated with 50 μΜ coronatine ("Cor") or 0.1% DMSO. GFP fluorescence was measured using a flow cytometer and was normalized to the relative level of GFP mRNA in each sample in order to control for differences in vector copy number and expression levels. Notably, the normalized fluorescence level of GFP was not affected by the presence of CorR in the absence of coronatine, indicating lack of coronatine-independent degradation. Moreover, coronatine treatment led to GFP degradation in CorR-dependent manner. Error bars represent the standard deviation of two biological replicates. FIGs 10J-10K show that coronatine treatment does not affect proliferation in human ESCs. H9 mESCs expressing pJAZ NLS-GFP-OsJAZ ³³ were grown in the presence of 50 μΜ coronatine ("Cor") or 0.1% DMSO ("Con"). In FIG 10J, flow cytometry was used to validate coronatine-dependent GFP degradation after 1 day of treatment. Cells were counted daily and growth curves are plotted in FIG 10K. The experiment was performed in triplicate, and a two-tailed paired t-test was used to calculate statistical significance, p-values are presented for each time point. Error bars represent standard deviation of technical replicates. FIGs 10L-10M show that coronatine treatment does not affect global gene expression patterns in human ESCs. H9 mESCs expressing pJAZ NLS-GFP-OsJAZ33 were treated for 2 days with 50 μΜ coronatine or 0.1% DMSO and collected for mRNA-Seq analysis. Experiment was performed twice (RepA and RepB). FIG 10L is a pairwise Spearman analysis showing perfect correlation between all samples. FIG 10M show that only two genes {HIST1H4H and LINC01547) demonstrated differential expression between coronatine and control samples. As controls, Fragments Per Kilobase of transcript per Million mapped reads values ("FPKM") are listed for Brachyury (7) and OCT4 (POU5F1), representing a differentiation marker (not expressed in hESCs) and a pluripotency marker (highly expressed in hESCS), respectively. These data suggest that HIST1H4H and LINC01547 are expressed at very low levels, and are unlikely significant to hESCs biology. Moreover, when the same search criteria were applied to identify genes that are differentially regulated between RepA and RepB (regardless of coronatine treatment), 7 genes were identified. This suggests that the two genes differentially expressed following coronatine treatment do not represent a significant transcriptional response, and are likely a result of inherent experimental noise.

[0023] FIGs 1 1 A-l 1G illustrate a coronatine-induced degradation rescue system. FIG

1 1 A shows the percent degradation in HEK-293T cells infected with the indicated pJAZ versions, selected and treated for 1 day with coronatine. GFP fluorescence was measured by flow cytometry and % degradation was calculated. The corresponding components and biological replicate number (n) are indicated. The bar for pJAZ 6-Os23 is in grey color, to indicate reduced GFP fluorescence in non-treated cells (FIG 10F). FIG 1 IB shows the relative median fluorescence of HEK-293T cells infected with pRAIDRS NLS-GFP-AID ⁴⁷ or pJAZ (version 7) NLS-GFP-OsJAZ ³³ , treated with the indicated concentrations of the corresponding hormone for 24 hours (left panel) or with 50 μΜ of hormone for the indicated time periods (right panel). GFP fluorescence was measured using flow cytometry. The experiment was repeated three times, and representative results are presented. In FIGs 1 1C- 1 ID, H9 hESCs were infected with pJAZ NLS-GFP-OsJAZ ³³ ("GFP-J") or pJAZ OsJAZ ³³ - p53 ("J-p53") that harbors an shRNA targeting the 3 '-UTR of p53 and an OsJAZ ³³ degron- fused p53 coding sequence lacking UTRs. Selected hESCs were treated with 50 μΜ coronatine ("Cor") or 0.1% DMSO ("Con") for 1 day. FIG 1 1 C shows western blot analysis (upper panel) and protein level quantification (lower panel) demonstrating knockdown of endogenous p53 (*) and expression of J-p53 (**), as well as effective (90%) coronatine- dependent degradation of J-p53. FIG 1 ID shows quantitative real-time PCR analysis for p53 target genes. Error bars represent the standard deviation of three technical replicates. P- values were calculated using unpaired Student' s t-test. The experiment was repeated twice, and representative results are displayed. In FIGs 1 lE-1 1 G, H9 hESCs were infected with pJAZ NLS-GFP-OsJAZ ³³ (harboring PuroR) and pRAIDRS NLS-mOrange-AID ⁴⁷ (harboring BSD), selected and cloned. Cells were treated with either ethanol and DMSO ("Con"), auxin and DMSO ("IAA"), ethanol and coronatine ("Cor"), or auxin and coronatine ("IAA+Cor"). After 24 hours, microscopic bright-field ("BF") and fluorescence images were taken (FIG 1 IE, scale bars, 100 μηι) and cells were subj ected to flow analysis (FIG 1 IF, contour plots; FIG 1 1G, quantification, error bars represent standard deviation.). Parental cells ("Par") are presented as autofluorescence control. Experiment was repeated three times and

representative results are displayed. See also FIGs 10A-10M, 12A-12G, and 15.

[0024] FIGs 12A-12G show that pRAIDRS and pJAZ function independently and simultaneously in multiple cell types. P19 mouse embryonal carcinoma cells (FIGs 12A-12B), NCI-H1299 human lung adenocarcinoma cells (FIGs 12C-12D) and HEK-293T human embryonic kidney cells (FIGs 12E-12F) were infected with pJAZ NLS-GFP-OsJAZ ³³ (harboring PuroR) and pRAIDRS LSmOrange-AID47 (harboring BSD) and selected with puromycin and blasticidin. Cells were treated with either EtOH and DMSO ("Con"), auxin and DMSO ("IAA"), EtOH and coronatine ("Cor"), or auxin and coronatine ("IAA+Cor"). After 24 hours, bright- field ("BF") and fluorescence microscope images were taken (FIGs 12 A, 12C, and 12E) and cells were subjected to flow cytometric fluorescence analysis (FIGs 12B, 12D, and 12F, contour plots on the left; quantification on the right). Parental non-infected cells ("Par") for each cell type are presented as background auto-fluorescence controls. In FIG 12G, the indicated cell types were infected, selected, and treated as above. Cells were subjected to flow cytometric analysis and quantification of median fluorescence levels is presented.

[0025] FIGs 13A-13E illustrate a dual molecular switch to dissect NOTCH1 Function in hESCs. In FIGs 13A-13D, H9 hESCs harboring pRAIDRS NLS-GFP-AID ⁴⁷ ("GFP-A") or pRAIDRS NICD-AID ⁴⁷ ("NICD-A") were maintained in mTeSRTMl media in the presence of 250 μΜ IAA. Cells were then washed twice and incubated for 3-5 days in the presence of 250 μΜ IAA (+) or 0.05% EtOH (-). FIG 13A is a western blot analysis demonstrating the knockdown of the full-length NOTCH1 receptor (detected with the anti-NICD antibody as a protein migrating between 200 and 250 kDa) in pRAIDRS NICD cells and the accumulation of GFP-AID ⁴⁷ following auxin removal. The diagonal line on the right side of the GFP blot was caused by a nick in the membrane. The accumulation of NICD-AID ⁴⁷ following auxin removal is displayed in FIG 14A. a- Tubulin serves as a loading control. Un-cropped blots are displayed in FIG 15. The experiment was performed twice, and representative results are displayed. FIG 13B show the relative expression of endogenous NOTCH1 in pRAIDRS NICD-A cells. QRT- PCR performed with primers that amplify only the endogenous NOTCH 1 gene demonstrates its knockdown at the mRNA level in pRAIDRS NICD-A cells. Experiment was performed 3 times and representative results are displayed. Error bars represent the standard deviation of 3 technical replicates. FIG 13C shows bright field ("BF") and GFP fluorescence microscopic images, as well as microscope images of alkaline phosphatase ("AP") -assayed cells,

demonstrating loss of ESC morphology and AP activity in pRAIDRS NICD-A cells in the absence of auxin. 4X digitally-magnified images of the outlined areas are presented as well. Scale bars, 100 μπι. FIG 13D shows QRT-PCR analysis of selected differentiation markers, as well as of the known NOTCH1 target HEY1. Results demonstrate the induction of

differentiation in pRAIDRS NICD-A cells in the absence of auxin. The experiment was performed 3 times, and representative results are displayed. Error bars represent standard deviation of 3 technical replicates. FIG 13E shows a heat map. H9 hESCs with the indicated pRAIDRS and pJAZ constructs were cultured for 4 days with mTeSRTM-E8TM, which contains FGF2 and TGF , or mTeSRTM-E6 media, which lacks FGF2 and TGF , and treated with 250 μΜ IAA and 50 μΜ coronatine, where indicated. QRT-PCR analysis was performed for selected genes and GAPDH-normalized values are represented as a heatmap. Two biological replicates are displayed, with the gene symbols corresponding to each repeat colored black and red. For each biological replicate, QRT-PCR analysis was performed in triplicates and heatmap represent average values. For cells harboring pRAIDRS NICD-A and pJAZ dnMl- GFP-J, white rectangles mark instances where dnMAMLl -GFP-J attenuated NICD-dependent activity by at least 2 fold (compared with the expression value in pRAIDRS NICD-A pJAZ GFP-J cells under the same condition). White circles indicate instances where coronatine treatment restored NICD-A-dependent activation by at least 2 fold.

[0026] FIGs 14A-14C illustrate a dual switch for dissection of NOTCH1 function in hESCs. H9 hESCs were infected with pRAIDRS harboring an shRNA targeting the full-length NOTCH1 receptor and an NICD-AID ⁴⁷ CDS ("NICD-A"). As a control, cells were infected with pRAIDRS NLS-GFP-AID ⁴⁷ ("GFP-A"). Cells were maintained with 250 μΜ auxin to prevent NICD-A accumulation. Following selection and isolation of colonies with ESC morphology, cells were infected with pJAZ dnMAMLl -NLS-GFP-OsJAZ ³³ ("dnMl -GFP-J") or pJAZ NLS- GFP-OsJAZ ³³ ("GFP-J") as control and post-selection colonies were expanded. FIG 14A is a western blot analysis of pRAIDRS GFP-A and NICD-A hESCs maintained in the presence of 250 μΜ auxin ("IAA"), washed and incubated for 4 h in the presence (+) or absence (-) of auxin. The western blot demonstrates NICD-A accumulation following auxin removal, a- Tubulin serves as a loading control. The experiment was repeated twice, and a representative blot is displayed. FIG 14B shows images of hESCs harboring pRAIDRS NICD-A and pJAZ dnMl- GFP-J were treated with 50 μΜ coronatine (+) or 0.1% DMSO (-) for 1 day. Bright-field ("BF") and fluorescence microscopic images demonstrate effective coronatine-dependent degradation of dnMl -GFP-J. Scale bars, 100 μτη. Dashed lines mark colony borders. The experiment was conducted more than three times, and representative images are displayed. FIG 14C shows the relative expression of hESCs harboring pRAIDRS NICD-A or GFP-A and pJAZ GFP-J or dnMl -GFP-J cultured for 4 days with TeSRTM-E8TM, which contains FGF2 and TGFp, or TeSRTM-E6 media, which lacks FGF2 and TGFp, and treated with 250 μΜ auxin and 50 μΜ coronatine where indicated. Quantitative real-time PCR analysis was performed for selected genes and GAPDH-normalized values are displayed (error bars represent standard deviation of three technical replicates). Dashed lines indicate instances where coronatine-mediated dnMl- GFP-J degradation restored NICD-A-dependent activity by at least twofold. The experiment was repeated three times, and representative results are displayed. See also FIGs 13A-13E and 15.

[0027] FIGs 15A-15H are images of un-cropped immunoblots from FIG 3 A (FIG 15 A),

FIG 5A (FIG 15B), FIG 5E (FIG 15C), FIG 9A (FIG 15D), FIG 11C (FIG 15E), FIG 14A (FIG 15F), FIG 10E (FIG 15G), and FIG 13 A (FIG 15H). Numbers on the left of each blot represent molecular weight in KDa.

[0028] FIG 16 shows a two-step cloning protocol. The strategy for constructing pRAIDRS/pJAZ rescue system plasmids is shown. In the first step, an empty pRAIDRS/pJAZ plasmid (Box 1) is restricted with Agel+EcoRI (step i-i), purified and ligated with a small hairpin dsDNA duplex containing Agel and EcoRI overhangs (step i-ii), which will give rise to the shRNA. In the second step, the POI's CDS is fused upstream or downstream of the degron, generating a POI-degron or degron-POI fusion, respectively. For example, to generate a POI- degron fusion, the shRNA-harboring plasmid (Box 2) is restricted with any desired combination of one or two REs from the 5'-MCS (e.g., Nhel+XmaT) (step ii-i), purified, and ligated (step ii-ii) with the POI's CDS, which was previously PCR-amplified using primers containing Nhel+Xmal sites, and restricted with these Nhel+Xmal. Inlet boxes depict the post-processing components harbored by each version. All elements expressed from the PGK-l/EFl promoter, including the RE sites, are in-frame. Hence, the cloned CDS must not include a STOP codon or any frame-shifting elements. The size of an empty pRAIDRS/pJAZ is ~9 kb (~6 kb between LTRs). Cloning extremely long CDSs may result in oversized viral genomes, which can hinder packaging and infection. Vectors harboring CDSs of 2 kb were sufficiently infectible. Testing multiple shRNA sequences before cloning the CDS is recommended. If possible, the shRNA should target the gene-of-interest' s UTRs to avoid targeting of the exogenous CDS. If this is impossible, the exogenous CDS should contain 3-4 synonymous mutations in the central shRNA binding region in order to avoid targeting by the shRNA, as described by Lee et al. (Lee et al., "Combining Competition Assays with Genetic Complementation Strategies to Dissect Mouse Embryonic Stem Cell Self-Renewal and Pluripotency," Nat. Protoc. 7:729-748 (2012), which is hereby incorporated by reference in its entirety) Pstl is not unique in vectors containing pEFla or OsCOIlB ^LRR

DETAILED DESCRIPTION OF THE INVENTION

[0029] In plants, auxin (indole-3 -acetic acid; "IA"A) induces degradation of a family of short-lived transcriptional repressors, the IAA proteins, by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of an F-box protein, TIRl (Hayashi et al., "The Interaction And Integration of Auxin Signaling Components," Plant Cell Physiol. 53 : 965-975 (2012), which is hereby incorporated by reference in its entirety).

Productive interaction of IAA proteins and TIRl in the presence of auxin leads to ubiquitynation of the target by recruitment of a Skpl-Cullin-F box protein ("SCF") ubiquitin E3 ligase complex, followed by proteasomal degradation. The SCF complex is highly conserved among eukaryotes (Zimmerman et al., "Structural Assembly of Cullin-RF G Ubiquitin Ligase Complexes," Curr. Opin. Struct. Biol. 20: 714-721 (2010), which is hereby incorporated by reference in its entirety), such that constitutive expression of TIRl allows a reconstitution of the auxin induced

degradation system in vertebrate cells. Due to the lack of an auxin-responsive system in animals, the hormone as well as the F-box protein are otherwise biologically silent and cause no measurable physiological changes in the absence of a target, thus minimizing possible side- effects of the treatment (Nishimura et al., "An Auxin-Based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6: 917-922 (2009), which is hereby incorporated by reference in its entirety). The role of auxin signaling through the F-box protein TIR-1 is analogous to the role of COI1 -mediated j asm onate ZEVI-domain ("JAZ") degradation in jasmonic acid ("JA") signaling.

[0030] The phytohormone JA and its metabolites regulate a wide spectrum of plant physiology, participating in normal development and growth processes as well as defense responses to environmental and pathogenic stressors. JA is activated upon specific conjugation to the amino acid L-isoleucine, which produces the highly bioactive hormonal signal (3R,7S)- jasmonyl-L-isoleucine ("JA-Ue"). Coronatine ("COR") is a Pseudomonas syringae virulence factor that structurally mimics JA-De.

[0031] The discovery of coronatine-insensitive mutants enabled the identification of COI1 as a key player in the JA pathway. Arabidopsis COI1 is an F-box protein that functions as the substrate-recruiting module of the SCF ubiquitin E3 ligase complex. Like other E3 ligases, SCF is involved in the ubiquitination of proteins, which targets the proteins for subsequent degradation by the 26S proteasome.

[0032] Arabidopsis JAZ proteins such as JAZ1, JAZ6, JAZ7, and JAZ 8 are SCF ^C01 substrate targets that associate with COI1 in a hormone-dependent manner. In the absence of hormone signal, the JAZ proteins actively repress the transcription factor MYC2, which binds to cis-acting elements of jasmonate-response genes. In response to cues that upregulate JA-Ile synthesis, the hormone stimulates the specific binding of JAZ proteins to COI1, leading to poly- ubiquitynation and subsequent degradation of the JAZ proteins by the 26S proteasome. JAZ degradation relieves repression of MYC2 and probably other transcription factors, permitting the expression of JA-responsive genes.

[0033] The present invention relates to a system for F-box hormone receptor regulated protein expression. The system includes a silencing nucleic acid molecule comprising a first promoter and an shRNA operably linked to the first promoter, where the shRNA silences expression of a target protein. The system also includes an expression nucleic acid molecule comprising a second promoter, an F-box hormone receptor operably linked to the second promoter, and a nucleic acid molecule encoding a fusion protein comprising a degron fused to the target protein, where the nucleic acid molecule encoding the fusion protein is operably linked to the second promoter.

[0034] As used herein, the term "promoter" refers to an untranslated DNA sequence usually located upstream of a coding region, which contains the binding site for RNA

polymerase and initiates transcription of the gene downstream of the promoter into mRNA. The promoters of the present invention may be constitutive promoters, which constitutively induce the expression of a target gene. The promoters of the present invention may also be inducible promoters, which induce the expression of a target gene at a specific site and a specific time.

[0035] In one embodiment, the first promoter is an RNA polymerase III promoter.

Exemplary RNA polymerase III promoters include, without limitation, U6 and HI promoters. In some embodiments, the RNA polymerase III promoter is a U6 promoter.

[0036] In one embodiment, the second promoter is an RNA polymerase II promoter. The

RNA polymerase II promoter may be a non-viral promoter. Exemplary non-viral RNA polymerase II promoters include, without limitation, phosphoglycerate kinase- 1 ("PGK-1) and elongation factor la ("EFla") promoters.

[0037] In another embodiment, the second promoter is a mammalian promoter. Suitable mammalian promoters include, without limitation, human, murine, bovine, canine, feline, ovine, porcine, ursine, and simian promoters. In one embodiment, the promoter is a human promoter.

[0038] The system of the present invention enables the genetic silencing of an endogenous protein of interest and its replacement with a degron-fused target protein in a mammalian cell. Silencing of the endogenous protein of interest is achieved through the expression of short hairpin RNA from the silencing nucleic acid molecule.

[0039] As used herein, the term "short hairpin RNA" or "shRNA" refers to an RNA molecule that leads to the degradation of mRNAs in a sequence-specific manner dependent upon complimentary binding of the target mRNA. shRNA-mediated gene silencing is well known in the art (see, e.g., Moore et al., "Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown," Methods Mol. Biol. 629: 141-158 (2010), which is hereby incorporated by reference in its entirety).

[0040] As used herein, the term "target protein" refers to a protein of interest. The silencing nucleic acid molecule of the present invention enables the silencing of an endogenous protein of interest. The expression nucleic acid molecule of the present invention enables the expression of a protein of interest whose degradation is induced by a molecule that binds to an F- box hormone receptor. Proteins of interest include all cellular proteins, including, but not limited to, proteins involved in embryonic stem cell division, proliferation, and differentiation.

[0041] The shRNA may target the 5'-untranslated region ("5'-UTR"), the coding region, or the 3 '-untranslated region ("3'-UTR") of a target protein. In one embodiment, the shRNA targets the 3'-UTR of the target protein. In another embodiment, the shRNA targets the 5'-UTR of the target protein. In yet another embodiment, the shRNA targets the coding region of the target protein.

[0042] In another embodiment, the shRNA targets a coding region of the target protein. In accordance with this embodiment, the fusion protein is resistant to silencing by the shRNA. For example, the nucleic acid molecule encoding the fusion protein may comprise silent mutations in the coding region of the target protein recognized by the shRNA {see, e.g., Jiang Y et al., "Rescue of the TTF2 Knockdown Phenotype with an siRNA-Resistant Replacement Vector," Cell Cycle 3 : 1151-1153 (2004) and Kim et al., "Coupling of RNAi-Mediated Target Downregulation with Gene Replacement," Antisense Nucleic Acid Drug Dev . 13 : 151-155 (2003), which are hereby incorporated by reference in their entirety).

[0043] As used herein, the term "F-box hormone receptor" refers to a plant protein or peptide containing at least one F-box domain that mediates the degradation of a degron-fused target protein in the presence of its respective hormone. The F-box hormone receptor may be codon-optimized for expression in mammalian cells. F-box hormone receptors are well known in the art (see, e.g., Spartz et al., "Plant Hormone Receptors: New Perceptions," Genes Dev. 22(16): 2139-2148 (2008), which is hereby incorporated by reference in its entirety).

[0044] In one embodiment, the F-box hormone receptor is selected from the group consisting of an auxin receptor or a jasmonate-isoleucine receptor. In another embodiment, the F-box hormone receptor is an auxin receptor. The auxin receptor may be Transport Inhibitor

Response 1 ("TIRl"). Exemplary TIRl receptors include, without limitation, Oryza sativa TIRl ("OfTOU"; NP 001052659; SEQ ID NO: 11), Arabidopsis thaliana TIRl, Arabidopsis lyrata TIRl, and TIRl homologs from other plant species, e.g., grape, tomato, corn, rubber tree, pea, wild tobacco, soybean, sorghum, or wheat. In one embodiment, the TIRl receptor is OsTTRl . In accordance with this embodiment, the OsTTRl receptor is a codon-optimized for expression in mammalian cells.

[0045] When the OsTIRl receptor is a codon-optimized for expression in mammalian cells, the expression nucleic acid molecule may comprise SEQ ID NO: 12.

[0046] In another embodiment, the F-box hormone receptor is a jasmonate-isoleucine receptor. The jasmonate-isoleucine receptor may be Coronatine Insensitive 1 ("COH").

Exemplary COI1 receptors include, without limitation, Arabidopsis thaliana COI1 (" 4tCOH"; NP 565919; SEQ ID NO: 10), Arabidopsis lyrata COI1, Oryza sativa COI1B ("OsCOIlB"; NP 001055700), and COI1 homologs from other plant species, e.g. , grape, tomato, corn, rubber tree, pea, wild tobacco, soybean, sorghum, or wheat. In one embodiment, the COI1 receptor is ^tCOIl .

[0047] As used herein, the term "chimeric protein" refers to a protein or polypeptide comprising two or more heterologous domains which are not found in the same relationship to one another in nature. The F-box hormone receptor of the present invention may be a chimeric F-box hormone receptor. Exemplary chimeric F-box hormone receptors may comprise amino acids sequences from Oryza sativa, Arabidopsis thaliana, Arabidopsis lyrata, or any other plant species having orthologous F-box hormone receptors.

[0048] Chimeric F-box hormone receptors may also comprise amino acid sequences from human F-box hormone receptors including, but not limited to, SKP2 (SEQ ID NO: 9).

[0049] In one embodiment, the chimeric F-box hormone receptor comprises amino acids

2-39 of SEQ ID NO: 1 1 coupled to amino acids 59-597 of SEQ ID NO: 13 (OsCOIB;

NP 001055700.1). In accordance with this embodiment, the chimeric F-box hormone receptor has the amino acid sequence of SEQ ID NO: 14 (OsTIR ^F"box - OsCOIlB ^LRR ):

MYPYDVPDYATYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIE RLSRKHVTVPFCYAASPAHLLARFPRLESLAVKGKPRAAMYGLIPEDWGAYARPWVAE L A APLECLK ALIILRRM V VTDDDL A AL VRARGFEVILQELKLDKC S GF S TD ALRL V ARS C RSLRTLFLEECSIADNGTEWLHDLAVNNPVLETLNFHMTELTVVPADLELLAKKCKSLIS LKISDCDFSDLIGFFRMAASLQEFAGGAFIEQGELTKYGNVKFPSRLCSLGLTYMGTNEM PIIFPFSALLKKLDLQYTFLTTEDHCQLIAKCPNLLVLAVRNVIGDRGLGVVADTCKKLQ RLRVERGDDDPGLQEEQGGVSQ VGLTTVAVGCRELEYIAAYVSDITNGALESIGTFCKN LCDFRLVLLDREERITDLPLDNGVRALLRGCTKLRRFALYLRPGGLSDTGLGYIGQYSGI I QYMLLGNVGETDDGLIRFALGCENLRKLELRSCCFSEQALARAIRSMPSLRYVWVQGY KASKTGHDLMLMARPFWNIEFTPPSSENANRMREDGEPCVDSQAQILAYYSLAGKRSD CPRSVVPLYPA. The amino acid sequence of OsTIR ^F"box - OsCOIlB ^LRR comprises a start codon (amino acid 1 of SEQ ID NO: 13), an HA Tag (amino acids 2-9 of SEQ ID NO: 13), and amino acids 2-39 of SEQ ID NO: 11 coupled to amino acids 59-597 of SEQ ID NO: 13.

[0050] As used herein, the term "degron" refers to a signal that targets a protein to a ubiquitin ligase complex in a hormone-dependent manner. Exemplary degrons include, without limitation, auxin-induced degradation ("AID") degrons and jasmonate ZIM-domain ("JAZ") degrons. The degrons of the present invention may comprise a full length wild type degron sequence or a portion of a full length wild-type sequence. In some embodiments, the degron is codon-optimized for expression in mammalian cells.

[0051] When the auxin receptor is Transport Inhibitor Response 1 ("TIR"), the degron is an auxin-induced degradation ("AID") degron. Exemplary AID degrons include, without limitation, Arabidopsis thaliana IAA14 ( _^ 4tIAA14; SEQ ID NO: 1), Arabidopsis thaliana IAA7 (AtlAAl; SEQ ID NO: 2), Arabidopsis thaliana IAA17 (_4flAA14; SEQ ID NO: 3). In one embodiment, the AID degron comprises amino acids 63-109 of SEQ ID NO: 3 (AID ⁴⁷ ). In another embodiment, the AID degron consists of amino acids 63-109 of SEQ ID NO: 3 (AID ⁴⁷ ). In accordance with this embodiment, AID ⁴⁷ is codon optimized for expression in mammalian cells.

[0052] When the jasmonate-isoleucine receptor is Coronatine Insensitive 1 ("COH"), the degron is a jasmonate ZIM-domain ("JAZ") degron. Exemplary JAZ degrons include, without limitation, Oryza sativa JAZ1 (OsJAZl; SEQ ID NO: 4), Oryza sativa JAZ2 (OsJAZ2; SEQ ID NO: 5), Oryza sativa JAZ 3 (OsJAZ3; SEQ ID NO: 7), Oryza sativa JAZ7 (OsJAZl; SEQ ID NO: 6), and Arabidopsis thaliana JAZ1 (AtJAZl SEQ ID NO: 8). The JAZ degron may comprise amino acids 109-141 or 104-146 of SEQ ID NO: 4. In one embodiment, the JAZ degron consists of SEQ ID NO: 15 (OsJAZ ³³ ):

HAAALPEMPIARKASLQRFLQKRKHRITTTSEP. In yet another embodiment, the JAZ degron consists of SEQ ID NO : 16 (OsJAZ ⁴³ ): PPQPAHAAALPEMPIARKASLQ

RFLQKRKHRITTTSEPYKKAA.

[0053] As used herein, the term "nuclear localization signal" or "NLS" refers to an amino acid sequence that directs import of a protein into the nucleus of the cell. The fusion protein of the present invention may further comprise a nuclear localization signal ("NLS"). When the degron is a JAZ degron, the fusion protein may further comprise a NLS. Nuclear localization signals are well known in the art and include, without limitation, the SV40 large T-antigen NLS (PKKKRKV; SEQ ID NO: 17). [0054] In one embodiment, the fusion protein comprises a target protein fused to the amino terminus of the degron. In another embodiment, the fusion protein comprises a target protein fused the carboxyl-terminus of the degron.

[0055] In some embodiments, the expression nucleic acid molecule further encodes a selectable marker operably linked to the second promoter. Suitable selectable markers include, without limitation, puromycin N-acetyl -transferase ("PAC"), blasticidin-S deaminase ("BSD"), 3'-glycosyl phosphotransferase ("Neo"), and Hygromycin B phosphotransferase ("Hpt"). In one embodiment, the selectable marker is selected from the group consisting of puromycin N-acetyl- transferase and blasticidin-S deaminase.

[0056] The expression nucleic acid molecules of the present invention may further encode one or more self-cleaving peptides. The use of self-cleaving peptides in multi-gene expression systems ("MGES") is well known in the art and is discussed further in, e.g., Wang et al., "2A Self-Cleaving Peptide-Based Multi-Gene Expression System in the Silkworm Bombyx mori," Scientific Reports 5: 16273 (2015) and Kim et al., "High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice," PLoS ONE 6:el8556 (2011), which are hereby incorporated by reference in their entirety. Self- cleaving peptides enable the simultaneous expression of multiple proteins from a single nucleic acid molecule.

[0057] In one embodiment, the expression nucleic acid molecule further encodes: (i) a first self-cleaving peptide between the F-box hormone receptor and the fusion protein and (ii) a second self-cleaving peptide between the fusion protein and the selectable marker. The first and second self-cleaving peptides may comprise porcine teschovirus-1 2A peptides.

[0058] The present invention also relates to a vector comprising the system of the present application, where the silencing nucleic acid molecule is coupled directly or indirectly to the expression nucleic acid molecule. The vector may be a lentiviral vector. As described above, lentiviral vectors enable the delivery of an independent rescue system containing continuously- expressed shRNA and an shRNA-immune hormone-degradable protein of interest that rescues the phenotype exerted by the shRNA. In some embodiments, the present invention relates to a mammalian cell infected with the lentiviral vector of the present application.

[0059] As used herein, the term "embryonic stem cells" refers to cells derived from embryonic sources that can self-renew and differentiate into multiple lineages.

[0060] As used herein, the term "cell line" or "cell lines" refers to a population of cultured cells derived from an identified parental cell type. [0061] As used herein, the term "primary cell" or "primary culture" refers to a cell or a culture of cells that have been explanted directly from an organism, organ, or tissue. Primary cultures are typically neither transformed nor immortal.

[0062] In one embodiment, the mammalian cell is selected from the group consisting of embryonic stem cells, cell lines, and primary cells. Exemplary embryonic stem cells include, without limitation, cells from already established lines, embryo carcinoma cells, embryonic fibroblasts including murine embryonic cells, other embryo-like cells, cells of embryonic origin or cells derived from embryos, many of which are known in the art and available from the American Type Culture Collection, Manassas, VA 20110-2209, USA, and other sources.

Additional suitable cells include, without limitation, mouse and human induced-pluripotent cells, epiblast cells, epliblast-like cells, and primary fibroblasts.

[0063] The present invention further describes a method for F-box hormone receptor regulated protein degradation in a mammalian host cell. This method involves providing a mammalian host cell; introducing the system of the present application into the mammalian host cell to produce a transgenic mammalian host cell; culturing said transgenic mammalian host cell under conditions that result in (i) silencing expression of the target protein and (ii) expression of the fusion protein; and contacting said transgenic mammalian host cell with a molecule that binds the F-box hormone receptor so that said fusion protein undergoes degradation.

[0064] The silenced target protein may be endogenous to the mammalian host cell. As described above, the mammalian host cell may be selected from the group consisting of embryonic stem cells, cell lines, and primary cells.

[0065] In one embodiment, the F-box hormone receptor is Transport Inhibitor Response

1 ("TIR1"), the degron comprises amino acids 63-109 of SEQ ID NO: 3 ("AID ⁴⁷ "), and the molecule that binds the F-box hormone receptor is auxin (Indole-3 -acetic acid, "IAA").

[0066] In another embodiment, the F-box hormone receptor is Transport Inhibitor

Response 1 ("TIR1"), the degron comprises amino acids 63-109 of SEQ ID NO: 3 ("AID ⁴⁷ "), and the molecule that binds the F-box hormone receptor is 1-Naphthaleneacetic acid ("NAA").

[0067] The degron may consist of amino acids 63-109 of SEQ ID NO: 3 ("AID ⁴⁷ ").

[0068] In one embodiment, the F-box hormone receptor has the amino acid sequence of OsTIR ^F"box -asCOIlB ^LRR (SEQ ID NO: 14), the degron comprises amino acids 109-141 of SEQ ID NO: 4, and the molecule that binds the F-box hormone receptor is coronatine. The degron may consist of SEQ ID NO: 15 (OsJAZ ³³ ) or SEQ ID NO: 16 (OsJAZ ⁴³ ). When the degron comprises a JAZ degron, the fusion protein may further comprises a nuclear localization signal. [0069] In a further embodiment, the silencing nucleic acid molecule is coupled directly or indirectly to the expression nucleic acid molecule within a vector.

[0070] In one embodiment, the introducing is carried out by infecting the mammalian cell with a vector. The vector may be a lentiviral vector.

[0071] The present invention also describes a method for F-box hormone receptor regulated target protein degradation in a mammalian host cell. This method involves providing a mammalian host cell and infecting, into the mammalian host cell, a first lentiviral vector comprising (i) a first silencing nucleic acid molecule comprising a primary first promoter and a first shRNA operably linked to the primary first promoter, where the first shRNA silences expression of a first target protein and (ii) a first expression nucleic acid molecule comprising a primary second promoter, a Transport Inhibitor Response 1 ("TIRl") receptor operably linked to the primary second promoter, and a nucleic acid molecule encoding a first fusion protein comprising an auxin-induced degradation ("AID") degron fused to a first target protein, where the nucleic acid molecule encoding the first fusion protein is operably linked to the primary second promoter. The method further involves infecting, into the mammalian host cell, a second lentiviral vector comprising (i) a second silencing nucleic acid molecule comprising a secondary first promoter and a second shRNA operably linked to the secondary first promoter, where the second shRNA silences expression of a second target protein and (ii) a second expression nucleic acid molecule comprising a secondary second promoter, a Coronatine Insensitive 1 ("COH") receptor operably linked to the secondary second promoter, and a nucleic acid molecule encoding a second fusion protein comprising a JAZ degron fused to a second target protein, where the nucleic acid molecule encoding the second fusion protein is operably linked to the secondary second promoter. The method further involves culturing the infected mammalian host cell under conditions that result in (i) silencing expression of the first and second target proteins and (ii) expression of the first and second fusion proteins; contacting the infected mammalian host cell with a molecule that binds the TIRl receptor so that said first fusion protein undergoes degradation; and contacting the infected mammalian host cell with a molecule that binds the COI1 receptor so that said second fusion protein undergoes degradation.

[0072] The first and second silenced target proteins may be endogenous to the mammalian host cell. As described above, the mammalian host cell may be selected from the group consisting of embryonic stem cells, cell lines, and primary cells.

[0073] In one embodiment, the first expression nucleic acid molecule further encodes a selectable marker operably linked to the primary second promoter and (ii) the second expression nucleic acid molecule further encodes a selectable marker operably linked to the secondary second promoter. As described above, the selectable marker may be selected from the group consisting of puromycin N-acetyl -transferase ("PAC") and blasticidin-S deaminase ("BSD").

[0074] As described above, the nucleic acid molecules of the present invention may further encode one or more self-cleaving peptides. When the first and second expression nucleic acid molecules further encode a selectable marker, the first and second nucleic acid molecules may also encode (i) a first self-cleaving peptide between the F-box hormone receptor and the fusion protein and (ii) a second self-cleaving peptide between the fusion protein and the selectable marker. In one embodiment, the first and second self-cleaving peptides are porcine teschovirus-1 2 A peptides.

[0075] Infection of the first lentiviral vector and the second lentiviral vector may occur sequentially or simultaneously.

[0076] The infected mammalian host cell can be contacted with a molecule that binds

TIR1 receptor and with a molecule that binds the COI1 receptor sequentially or simultaneously.

[0077] In one embodiment, the AID degron consists of amino acid 63-109 of SEQ ID NO: 3.

[0078] In another embodiment, the COI1 receptor is OsTIRl ^F"box -OsCOIB 1 ^LRR (SEQ ID

NO: 14).

[0079] The JAZ degron may comprise amino acids 109-141 of SEQ ID NO: 4. The JAZ degron may also consist of SEQ ID NO: 15 (OsJAZ ³³ ) or SEQ ID NO: 16 (OsJAZ ⁴³ ). When the degron comprises a JAZ degron, the fusion protein may further comprises a nuclear localization signal.

EXAMPLES

Material and Methods for Examples 1-4

[0080] Cell culture. HEK-293T, HCT-116 and NUi/393 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Corning), 1 mM sodium pyruvate, 2 mM L- glutamine and PenStrep (all from Gibco). NCI-H358 and NCI-H1299 cells (obtained from the American Type Culture Collection) were cultured in RPMI-1640 (Cellgro) supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine and PenStrep. Validated, mycoplasma- free hESCs and mESCs were obtained from the Pluripotent Stem Cell Core Facility at ISMMS. ESCs were routinely monitored for ES-like morphology and expression of Nanog and Oct4 (Pou5fl) using quantitative real-time PCR. CCE and Rl mESCs, as well as P19 mouse embryonal carcinoma cells, were cultured in DMEM supplemented with 15% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, non-essential amino acids, PenStrep, 10 nM 2- mercaptoethanol and 100 U ml ^"1 LIF (ESGRO) on plates coated with 0.1% gelatin (Millipore, catalogue number ES-006-B). H9 hESCs were cultured with mTeSR™l (Stem Cell

Technologies) on plates coated with Matrigel (BD Biosciences, catalog number 354234). For controlling the presence of FGF2 and TGFp, TeSR™-E8™ and TeSR™-E6, which contain and lack FGF2/TGFb, respectively, were used. All cells were grown at 37°C in a humidified atmosphere of 5% C0 ₂ and passaged on average twice per week. All cells were tested negative for mycoplasma using the e-Myco Mycoplasma PCR Detection Kit (iNtRON). Where indicated, cell numbers were recorded with each passage and population doublings were calculated as Log ₂ (cell output/cell input).

[0081] Lentiviral infection and selection. For the production of lentiviral particles, lxlO ⁷

FIEK-293T cells were resuspended in growth media (as described above) and transfected with 20 mg lentiviral vector, 20 mg psPAX2 packaging plasmid and 10 mg pMD2.G envelope plasmid using the calcium phosphate method. Cells were then plated in a 10-cm dish and cultured for 1 day. On the second day, media were replaced and cells were incubated at 32°C. Viral supernatants were collected on the morning and evening of the third and fourth days, passed through a 0.22- or 0.45-mm cellulose acetate filter and concentrated ~25-fold using an Amicon Ultra- 15 Centrifugal Filter (Millipore). Cells were infected with concentrated virus diluted in their appropriate media in the presence of 8 mg ml ^"1 polybrene (Sigma) for -16 h at 37°C.

Selection was applied 2 days following infection with either 1-2 mg ml ^"1 Puromycin (Fisher

Scientific) or 10-20 mg ml ^"1 Blasticidin-S (Fisher Scientific). Where indicated, colonies (clones) of mESCs and hESCs with typical ESC morphology were manually isolated and expanded.

[0082] Chemicals and treatments. Auxin (IAA, Fisher Scientific, catalogue number

AC12216) was dissolved in ethanol to a final concentration of 500 mM. Cells were treated with 50 μΜ IAA or 0.01% ethanol as a control, unless otherwise indicated. Coronatine (Sigma, catalogue number C8115) was first dissolved in dimethylsulfoxide ("DMSO") to a concentration of 50 mM and then diluted in DMEM to a final concentration of 5 mM. Cells were treated with 50 μΜ coronatine or with 0.1% DMSO as a control, unless otherwise indicated. Aphidicolin (Fisher Scientific, catalogue number AC61197) was diluted in DMSO to a final concentration of 10 mM. Cells were treated with 1 μΜ aphidicolin or with 0.01% DMSO as a control, unless otherwise indicated. All trans-ret oic acid (Fisher Scientific, catalogue number 302-79-4) was dissolved in ethanol.

[0083] Staining. Stemgent's Alkaline Phosphatase staining kit (catalog number 00-009) was used according to the manufacturer's protocol. Crystal violet (CV) staining was performed by incubating cells for 5 minutes with CV solution (10 mM CV, 10% ethanol in water), followed by three to five gentle washes with water. For both AP and CV staining, plates were scanned using a standard desktop scanner and images were digitally adjusted for brightness and contrast. Acetic acid was used to extract CV, which was then quantified using a spectrophotometer at 590 nm. DAPI (4' ,6-diamidino-2-phenylindole) staining was performed by fixing cells (plated on cover slips) with 4% paraformaldehyde in PBS for 30 minutes, washing twice with PBS (for 5 minutes), treating with 0.2% Triton X-100 and 1% BSA in PBS for 30 min, washing with PBS and incubating with 0.2 mg ml ^"1 DAPI for 10 minutes. Cells were then washed once with PBS and mounted on microscope slides. Images acquired with a microscope were digitally adjusted for brightness and contrast. All images from the same experiment were processed identically.

[0084] Flow cytometry. Flow cytometry was performed on a BD LSRII machine. For

GFP and mOrange fluorescence analysis, cells were trypsinized, neutralized with FBS- containing media, supplemented with 0.2 mg ml ^"1 DAPI and kept on ice. Cells were gated on forward scatter area ("FSC-A") and side scatter area ("SSC-A"), on FSC width ("FSC-W") and FSC-A to eliminate cell aggregates, and on FSC-A and DAPI to eliminate dead cells.

GFP and mOrange fluorescence intensities were detected using the fluorescein isothiocyanate ("FITC") and DsRed channels, respectively. Background autofluorescence was measured using parental noninfected cells. Background-subtracted median fluorescence was normalized to the control -treated sample, to calculate relative median fluorescence. To calculate % degradation, relative median fluorescence was subtracted from 1. For measurement of apoptotic index, cells were collected by trypsinization together with all cells floating in the media, counted and 3xl0 ⁵ cells per sample were washed twice with PBS and stained using Annexin V:PE Apoptosis Detection Kit I (BD Biosciences, catalog number 559763) according to the manufacturer's protocol. Cells were gated on FSC-A and SSC-A, and on FSC-W and FSC-A to eliminate cell aggregates. 7-aminoactinomycin D ("7-AAD") was detected using the PerCP-Cy5.5 filter. Apoptotic index was calculated as the percentage of cells that are 7- AAD negative and Annexin V-Phycoerythrin positive. For measurement of mitotic index, cells were collected by trypsinization together with all cells floating in the media, neutralized with FBS-containing media, washed and fixed by slowly adding ice-cold 70% ethanol/Hank's balanced salt solution while vortexing. Cells were kept for at least 2 hours at -20°C, washed with PBS, incubated for 15 minutes on ice with 0.25% Triton X-100 in PBS and resuspended in 100 ml PBS-BA (PBS supplemented with 1% BSA and 0.02% sodium azide) containing 2 ml anti-Phospho-Histone H3 SerlO antibody (Cell Signaling, catalog number 9706). Cells were incubated for 2 hours at room temperature with gentle rocking, washed twice with PBS- BA, resuspended in 100 ml PBS-BA supplemented with Alexa Fluor 546 secondary antibody (1 :200, Life Technologies), incubated for 30 minutes at room temperature in the dark with gentle rocking, washed with PBS-BA, resuspended in 400 ml PBS containing 50 mg ml ^"1 RNAse-A and incubated 30 minutes at 37°C in the dark. Samples were then cooled, supplemented with DAPI to a final concentration of 2 mg ml ^"1 and incubated on ice for 15 minutes. Unstained and secondary-antibody only samples served as controls. For analysis of cell-surface SSEA-1 expression, cells were trypsinized, washed three times with PBS supplemented with 0.5% BSA (PBSB) and lxlO ⁵ cells were resuspended in 25 ml PBSB and 10 ml PE-conjugated anti-SSEA-1 antibody (R&D Systems, catalog number FAB2155P) or IgG-PE for isotype control, incubated 30 minutes on ice, washed twice with PBSB, filtered and supplemented with DAPI to a final concentration of 0.2 mg ml ^"1 .

[0085] Quantitative real-time PCR and expression heatmaps. Total RNA was extracted using TRIZOL (Ambion) and 1-2 μg were reverse transcribed using the High Capacity Reverse Transcription Kit (Life Technologies, catalog number 4368814) according to the manufacturer's protocol. QRT-PCR was performed in triplicates or quadruplicates using the Fast SYBR Green Master Mix (Life Technologies, catalog number 4385612) on a LightCycler480 Real-Time PCR System (Roche). Expression was calculated using the ACt method. Relative expression was calculated by dividing the average level of each gene to that of the housekeeping gene GAPDH measured in the same cDNA sample. Gene-specific primers are listed in Table 2. When data are displayed as bar charts, error bars represent the standard

Table 2. QRT-PCR Primers.

deviation of technical replicates. To generate gene expression heatmaps, normalized average expression levels were analysed using the Gene Cluster 3.0 Software (de Hoon et al., "Open Source Clustering Software," Bioinformatics 20: 1453-1454 (2004), which is hereby incorporated by reference in its entirety). Data were log transformed and genes were mean centered. Genes were then hierarchically clustered using uncentered correlation similarity metric and average linkage. [0086] Western blot analysis. Cells were lysed in RIPA-B buffer (20 mM Na ₂ HP0 ₄ pH 7.4, 150 mM NaCl and 1% Triton X-100) supplemented with Protease Inhibitor Cocktail (Roche) for 30 minutes on ice with occasional vortexing, followed by 30 minutes

centrifugation at 13,000 relative centrifugal force at 4°C. For CDK ^pY15 detection, lysis buffer was supplemented with 1 mM dithiothreitol, 50 mM NaF, 30 mM tetrasodium pyrophosphate, 0.1 mM sodium orthovanadate, 10 mM β-glycerophosphate, and 15 mM para- nitrophenylphosphate. The BCA Protein Assay Kit (Thermo Scientific, catalog number 23225) was used to determine protein concentration. Next, 20-75 μg protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad).

Membranes were blocked with TBST (10 mM Tris-HCl pH 7.9, 150 mM NaCl and 0.05% Tween-20) containing 3% skim milk, incubated with primary antibodies overnight, washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham), washed three times with TBST and subsequently reacted with ECL or ECL Prime (GE Healthcare). Luminescence was detected with X-ray films, which were scanned, or using the Bio-Rad ChemiDoc MP System. Blots were processed digitally by adjusting the brightness and contrast, and by rotating and cropping, when necessary. The following primary antibodies were used: rabbit anti-GFP (Invitrogen, catalog number A-6455, 1 :500), mouse anti-CHKl (FL-393, Santa Cruz Biotechnology, catalog number sc-8408, 1 : 1,000), mouse anti-P-actin (Sigma, catalog number A2066, 1 :4,000), rabbit anti-p53 (DO-1, Santa Cruz Biotechnology, catalog number sc-6243, 1 : 1,000), mouse anti-p53 (Santa Cruz Biotechnology, catalog number sc-126, 1 :500), rabbit anti-p21 (Santa Cruz Biotechnology, catalog number sc-397, 1 :500), rabbit anti-NANOG (Millipore, catalog number AB5731, 1 : 1,000), mouse anti-HA (Abeam, catalog number ab l6918, 1 :4,000), rabbit anti-phospho Cdc2 (CDK1) Tyrl 5 (Cell Signaling, catalogue number 91 11, 1 :500), mouse anti-CDC25A (Santa Cruz Biotechnology, catalog number sc-7389, 1 :250), sheep anti-Notch-1 Intracellular Domain (R&D Systems, catalog number AF3647, 1 :200), and mouse anti -a- Tubulin (Sigma, catalog number T9026, 1 : 1,500). Quantification of protein level was performed using the ImageJ software (Schneider et al., "NTH Image to ImageJ: 25 Years of Image Analysis," Nat. Methods 9:671-675 2012), which is hereby incorporated by reference in its entirety). Uncropped immunoblot scans are displayed in FIG 15.

[0087] mRNA-Seq. For testing the global transcriptional effect of coronatine treatment, H9 hESCs expressing pJAZ NLS-GFP-OsJAZ ³³ and pRAIDRS NLS-mOrange- AID ⁴⁷ were treated for 2 days with 50 μΜ coronatine (Cor) or 0.1% DMSO (Con). The experiment was repeated twice (replicates A and B). RNA was extracted with TRIZOL (Ambion). Sample preparation and sequencing was performed by Girihlet Inc. Briefly, total RNA was evaluated for quality and quantity using the Agilent RNA 6000 Nano Kit on an Agilent Bioanalyzer. Libraries were prepared using TruSeq RNA Library Prep Kit (Illumina). mRNA was isolated from 500 ng of total RNA using poly T beads and cDNA was synthesized using Superscript Reverse Transcriptase (ThermoFisher Scientific) and random primers. The cDNA ends were blunted, 'A' base added and adapters ligated. A total of 15 cycles of PCR were performed to generate cDNA libraries. The libraries' concentration was measured using an Agilent DNA 1000 Kit on an Agilent Bioanalyzer. Libraries were sequenced on a NextSeq 500 machine (Illumina) with 1 *75 bp reads.

[0088] Data analysis. The resulting fastq files were mapped to the human genome

(version hgl9) using the TopHat programme (with Bowtie2). The output .bam files were processed through the Cuffquant programme to generate normalized read counts. The resulting .cxb files were processed through the Cuffdiff programme to generate fragments per kilobase of transcript per million mapped reads (FPKM) values. Raw data (fastq files), as well as FPKM values, were uploaded to the GEO database (GSE74457). On average, there were 7.7>< 10 ⁷ reads per sample, which mapped to 23,622 human genes. Lowly expressed genes with an average FPKM value <0.1 were excluded, narrowing the total gene count to 15,928. The BRB-Array Tools software (Simon et al., "Analysis of Gene Expression Data Using BRB-Array Tools," Cancer Inform. 3 : 11-17 (2007), which is hereby incorporated by reference in its entirety) was used to calculate Spearman pairwise correlation between all samples (FIG 11L). To identify genes that were differentially regulated following coronatine treatment (FIG 10M), the gene list was filtered to include genes that meet the following criteria: (1) genes that scored a -value <0.05 in a two-tailed paired t-test comparing coronatine-treated samples with control samples; (2) genes that had a fold change >2 between coronatine and control samples in both replicates; and (3) coding genes and long non-protein- coding RNAs (excluding small RNAs). Using these criteria, only two genes demonstrated differential expression between coronatine and control samples. When the same criteria were applied to search for genes that were differentially regulated between the two biological replicates, seven such genes were identified.

[0089] Construction of pRAIDRS and pJAZ. Initially, pRAIDRS and pJAZ vectors were synthesized as a cassette containing the following components (restriction enzyme- binding sites, REBSs, are italicized): AscI/pVGK-l (partial sequencey^a/J/Kozak

Sequence/Hormone Receptor/£coRF/5 '-P2A/5'-MCS/Degron/3 '-MCS/3'-P2A/N5/7/Selectable Marker /Aatll...Kpnl. Cassettes were cloned using Ascl+Kpnl into an empty pLKO.1 -Puro lentiviral vector (Moffat et al., "A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-content Screen," Cell 124: 1283-1298 (2006), which is hereby incorporated by reference in its entirety). Different versions of the vectors were then constructed by shuffling components between existing versions or adding new components using restriction enzymes. Specifically, degrons were cloned using Xmal+Xbal, hormone receptors wit Sall+EcoRV and selectable markers with Pstl+Aatll. When indicated, restriction-free cloning (RFC) (Ulrich et al., "Exponential Megapriming PCR (EMP) Cloning— Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints," PLoS ONE 7:e53360 (2012), which is hereby incorporated by reference in its entirety) was used. Primers and shRNA sequences are listed in Tables 3 and 4, respectively.

able 3. Cloning Primers.

Table 4. shRNA Oli onucleotides

Sequences were codon-optimized (using the GeneArt algorithm) to increase their human Codon Adaptation Index ("CAI"), while avoiding the generation of any REBS that would render unique REBSs in the other parts of the vector non-unique. Components were designed and constructed as follows: OsTTRl is a codon-optimized (CAI=0.95): O. sativa (rice) TIR1 gene (encoding NP 001052659; SEQ ID NO: 1 1), excluding the STOP codon. ^tCOIl is a codon-optimized (CAI=0.96) ^. thaliana COI1 gene (encoding NP 565919; SEQ ID NO: 10), excluding the STOP codon. OsTIRl ^F"box -;4tCoi l ^LRR : a chimeric receptor composed of an OsTTKl F-box domain (Tan et al., "Mechanism of Auxin Perception by the TIR1 Ubiquitin Ligase," Nature 446:640-645 (2007), which is hereby incorporated by reference in its entirety) (amino acids 1-39) and an AtC ll leucine-rich repeat (Sheard et al., "Jasmonate Perception by Inositol -phosphate-potentiated COIl-JAZ Co-Receptor," Nature 468:400-405 (2010), which is hereby incorporated by reference in its entirety) (amino acids 52-592) was constructed using RFC. Megaprimers were generated using pRAIDRS as a template and primers 1+2. These megaprimers were used with pJAZ 1 as a template, to generate pJAZ 2. OsCOIlB is a codon- optimized (CAI=0.95) rice COI1B (encoding NP 001055700.1 ; SEQ ID NO: 13). OsTIRl ^{F" box} -OsCOIlB ^LRR (SEQ ID NO: 14): a chimeric receptor composed of a Met-HA-tagged OsTTKl F-box domain (Tan et al., "Mechanism of Auxin Perception by the TIRl Ubiquitin Ligase," Nature 446:640-645 (2007), which is hereby incorporated by reference in its entirety) (amino acids 2-39 of SEQ ID NO: 1 1) and an OsCOIlB leucine-rich repeat (Sheard et al., "Jasmonate Perception by Inositol-phosphate-potentiated COIl-JAZ Co-Receptor," Nature 468:400-405 (2010), which is hereby incorporated by reference in its entirety) (amino acids 59- 597 of SEQ ID NO: 13) was constructed using RFC. Megaprimers were generated using pJAZ 2 ^HA as a template and primers 11+12. These megaprimers were used with pJAZ 5-Os23 or 5-At23 as a template, to generate versions 6-Os23 or 6- _^ 4t23, respectively. 5'-P2A is a 2A peptide derived from porcine teschovirus-1. Codons were edited to achieve low degree (81%) of homology with the 3 '-P2A sequence, to reduce recombination likelihood. 5'-MCS: four tandemly arranged 6-bp REBSs (BstBI, Nhel/Bmtl, SnaBI, and XmallSmaT). AID ⁴⁷ is a codon-optimized (CAI=0.93) 47-amino acid segment that corresponds to amino acids 63-109 of A. thaliana IAA17 (AOAA17, NP_171921; SEQ ID NO: 2). AID ³³ (corresponding to AtlAAl? amino acids 63-95) was generated by PCR amplification using AID ⁴⁷ as a template and primers containing REBSs enabling replacement of the degron segment. At] AZ ²³ is a codon-optimized (CAI=0.96) 23-amino acid segment that corresponds to amino acids 199- 221 of A. thaliana JAZ1 (AtJAZl, NP_973862; SEQ ID NO: 8). ^tJAZ ³¹ is an extended version of ^tJAZ ²³ and was PCR-amplified with At] AZ as a template and primers 3+4, and then cloned with Xmal+Xbal. At] AZ ^FL is the full-length A. thaliana JAZ1 protein (non-codon optimized) and was PCR-amplified from a JAZ1 -containing plasmid (Sheard et al., "Jasmonate Perception by Inositol-phosphate-potentiated COIl-JAZ Co-Receptor," Nature 468:400-405 (2010), which is hereby incorporated by reference in its entirety) with primers 5+6, and cloned with Xmal+Xbal. OsJAZ ²³ is a codon-optimized (CAI=0.98) 23-amino acid segment corresponding to amino acids 1 14-136 of O. sativa JAZ1 (OsJAZl, NP 001064513; SEQ ID NO: 4). OsJAZ ³³ is an extended version of OsJAZ ²³ (corresponding to OsJAZ 1 amino acids 109-141) and was PCR amplified with OsJAZ ²³ as a template and primers 7+8, and cloned with Xmal+Xbal ^' . 3 '-MCS: four tandemly arranged 6-bp REBSs (Xbal, Hpal, BamHI and Pstl). It is noteworthy that Pstl is not unique in vectors containing pEFla or OsCOIlB ^LRR . 3 '-P2A is identical to 5'-P2A, except for different codon usage. PuroR is a codon-optimized (CAI=0.92) N-acetyltransferase gene. BSD is a codon-optimized (CAI=0.96) Blasticidin-S deaminase gene. HA tag: to generate pRAIDRS 7 ^HA , an HA tag (YPYDVPDYA), preceded by a methionine (Met), was inserted upstream of OsTIRl by cassette PCR amplification with pRAIDRS as the template and primers 9+10, and cloning this cassette into pRAIDRS with Sall+BstBI. To generate pJAZ 2 ^HA , a Met-preceded HA tag was cloned upstream of the OsTTRl ^F"box -,4tCOIl ^LRR by cassette PCR amplification with pJAZ 2 as the template and primers 9+10, and cloning this cassette into pJAZ with Sall+BstBI. GFP: enhanced GFP was PCR amplified from pLKO. l-Puro-IRES-GFP with primers 16+14 and cloned into pJAZ or pRAIDRS using Nhel+XmaL mOrange was PCR amplified from pFUW-mOrange with primers 17+15 and cloned into pJAZ or pRAIDRS with Nhel+XmaL NLS: an SV40 large T- antigen NLS (PKKKRKV; SEQ ID NO: 17) was fused to the amino terminus of GFP or mOrange by PCR amplifying an NLS-GFP cassette with primers 13+14 or an NLS-mOrange cassette with primers 13+15, and cloning into pJAZ or pRAIDRS with Nhel+XmaL HsSKP2 ^{F" box} -^tCOIl ^LRR : a chimeric receptor composed of an Homo sapiens SKP2 ("HsSKP2") F-box domain (amino acids 95-132 of SEQ ID NO: 9) and ^tCOIl leucine-rich repeat (Sheard et al., "Jasmonate Perception by Inositol-phosphate-potentiated COI1-JAZ Co-Receptor," Nature 468:400-405 (2010), which is hereby incorporated by reference in its entirety) (amino acids 50- 592 of SEQ ID NO: 10) was constructed using RFC. Megaprimers were generated using i¾SKP2-containing plasmid (Zheng et al., "Structure of the Cul l-Rbxl-Skpl-F boxSkp2 SCF Ubiquitin Ligase Complex," Nature 416:703-709 (2002), which is hereby incorporated by reference in its entirety) as a template and primers 18+19. These megaprimers were used with pJAZ 1 as a template to generate pJAZ 3. i¾SKPl - 4tCOIl ^LRR fusions: chimeric receptors composed of either full-length HsSKP l or an N-terminal -truncated HsSKPl lacking amino acids 1-129 (HsSKPl ^1"129 ) and either ^tTIRl ^F"box -^tCOIl ^LRR or just ^tCOIl ^LRR were constructed using RFC. Megaprimers were generated using pCDNA3.1 -SKPl -HA as a template and the following primer combinations: 20+21 for HsSKPl -OsTIRl ^F"box -,4tCOIl ^LRR , 20+22 for HsSKP l-,4tCOIl ^LRR , 20+23 for H5SKPl ^A1"129 -O5TIRl ^F"box -^tCOIl ^LRR and 20+24 for/¾SKP l ^"129 -,4tCOIl ^LRR Megaprimers were used with pJAZ 2 as a template to generate pJAZ 4a-d. pEF l a: human EF l a promoter was cloned using RFC: megaprimers were generated using pEF la-BirA-V5-His as a template and primers 25+26. These megaprimers were used to switch pPGK-1 into pEFl a in pRAIDRS and pJAZ. Site-directed mutagenesis (Zheng et al., "An Efficient One-Step Site-Directed and Site-Saturation Mutagenesis Protocol," Nucleic Acids Res. 32:el 15 (2004), which is hereby incorporated by reference in its entirety) was performed with primers 33+34 to eliminate the Agel site in pEF l a.

[0090] Construction of rescue systems. In general, rescue system vectors were constructed using the two-step cloning protocol (FIG 16). The specific components used were as follows: for pRAIDRS AID ⁴⁷ -NANOG (A-NANOG): an shRNA cassette targeting mouse Nanog 3 '-UTR was generated by annealing oligonucleotides 101+102, as previously described (Lee et al., "Combining Competition Assays with Genetic Complementation Strategies to

Dissect Mouse Embryonic Stem Cell Self-renewal and Pluripotency," Nat. Protoc. 7:729-748 (2012), which is hereby incorporated by reference in its entirety), and cloning into pRAIDRS with Agel+EcoRI. Mouse Nanog CDS was amplified from pCR4-Nanog using primers 27+28 and cloned into the shRN A- containing pRAIDRS with Xbal+BamHI. For pRAIDRS p53- AID ⁴⁷ (p53-A): mouse p53 CDS was amplified from pSIN-EF2-Myc-Trp53 using primers

29+30 and cloned into pRAIDRS with BstBI+Nhel. For pRAIDRS AID ⁴⁷ -CHK1 (A-CHK1): an shRNA cassette targeting mouse Chkl 3 '-UTR was generated by annealing

oligonucleotides 103+104 and cloning into pRAIDRS with Agel+EcoRI. Mouse Chkl CDS was amplified from pGEM-T-Chkl using primers 31+32 and cloned into pRAIDRS with Xbal+BamHI. For pRAIDRS NICD-AID ⁴⁷ (NICD-A): an shRNA cassette targeting human NOTCHl 3 '-UTR was generated by annealing oligonucleotides 105+ 106 and cloning into pRAIDRS with Agel+EcoRI. Human NICD CDS was amplified from EF.hICNl .Ubc.GFP (Addgene Plasmid 17626) using primers 37+38 and cloned into pRAIDRS with Nhel+SnaBI. For pJAZ OsJAZ ³³ -p53 (J-p53): an shRNA cassette targeting human TP 533 '-UTR was generated by annealing oligonucleotides 109+1 10 and cloning into pLKO. l Puro with Agel+EcoRI. Next, the cassette was transferred to pJAZ with EcoRI+Sphl. Human p53 CDS was amplified from pLenti6/V5-p53_wt p53 (Addgene Plasmid 22945) using primers 41+42 and cloned into pJAZ with Xbal+BamHI. For pJAZ dnM AML 1 -NL S -GFP - Os J AZ ³³ (dnMl - GFP-J): dominant-negative human MAMLl (dnMAMLl) (Yu et al., "Notch Signaling

Activation in Human Embryonic Stem Cells is Required for Embryonic, but not Trophoblastic, Lineage Commitment," Cell Stem Cell 2:461-471 (2008), which is hereby incorporated by reference in its entirety), corresponding to MAMLl AAs 13-74, was cloned from pHAGE-N- V5-MAML1 -FL (Addgene Plasmid 37048) using primers 39+40 and cloned into pJAZ NLS- GFP-OsJAZ ³³ with BstBI+Nhel.

Example 1 - pRAIDRS Functions as an Auxin-Induced Degradation Rescue System

[0091] Applicants aimed to design a vector that enables depletion of an endogenous gene-of-interest and its replacement by an exogenous POI whose degradation is induced by auxin. Such an approach represents a genetic complementation (rescue) system, in which a phenotype exerted by silencing a gene-of-interest is conditionally reversed by exogenous expression of that gene product. To this end, pRAIDRS (RNAi and auxin-induced

degradation rescue system), a lentiviral vector containing all elements for construction of an auxin-regulated rescue system was engineered. As depicted in FIG. IB, a U6 promoter drives the expression of an shRNA that silences an endogenous gene-of-interest. A second promoter, either phosphoglycerate kinase-1 ("pPGK- 1") or the stronger elongation factor l a ("pEF l a") (FIG 2A), followed by a Kozak sequence, drives the expression of an mRNA encoding three in-frame proteins separated by two porcine teschovirus-1 2 A (P2A) peptides. The first protein is a codon-optimized Oryza sativa (rice) TIRl auxin receptor (OsTIRl). The second component is a shortened AID degron derived from Arabidopsis thaliana IAA17 (" 4tIAA17"), which can be fused to either terminus of the POI. The last component is a selectable marker, either puromycin N-acetyl-transferase ("PuroR") or blasticidin-^ deaminase ("BSD"), conferring puromycin or blasticidin resistance, respectively. Mammalian cells transduced with pRAIDRS express OsTTRl , which associates with SKP1 and forms a functional SCF ^TIR1 complex (Nishimura et al., "An Auxin-based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009), which is hereby incorporated by reference in its entirety). Following auxin treatment, SCF ^TIR1 mediates degron polyubiquitination, leading to degradation of the POI (FIG 1 A).

[0092] The full-length ^tIAA17, originally used in pAID (Nishimura et al., "An Auxin- based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009), which is hereby incorporated by reference in its entirety), is imperfect as a degron due to its large size (228 amino acids), its propensity to confer nuclear localization (Arase et al., "IAA8 Involved in Lateral Root Formation Interacts with the TIRl Auxin Receptor and ARF Transcription Factors in Arabidopsis, "PLoS ONE 7:e43414 (2012), which is hereby incorporated by reference in its entirety) and other potentially undesirable activities it possesses as a plant transcription factor. Therefore, the minimal required AID degron was mapped to a 47-AA region ("AID ⁴⁷ ") spanning ^tlAAl 7 residues 63-109 (FIGs 2B-2C), which mostly overlaps with a previously reported shortened AID degron (Morawska et al., "An Expanded Tool Kit for the Auxin-inducible Degron System in Budding Yeast," Yeast 30:341-351

(2013), which is hereby incorporated by reference in its entirety). Notably, in pRAIDRS- transfected HEK-293T cells, green fluorescent protein ("GFP") spontaneously cleaved from the full-length _4ΛΑΑ17 degron ("AID ²²⁸ "), but not from AID ⁴⁷ (FIGs 2C-2D), suggesting that a shorter degron might also be more cleavage resistant. However, as other labs who have used AID ²²⁸ did not report spontaneous cleavage, this phenomenon might be cell line-specific, POI-specific or due to the vector architecture. The degradation of cytoplasmic and nuclear POIs was compared by analyzing the effect of a nuclear localization signal ("NLS") on the degradation of GFP-AID ⁴⁷ . The use of a NLS was found to be highly effective, but resulted in faster NLS-GFP-AID ⁴⁷ degradation (FIGs 2E-2F). Example 2 - pRAIDRS Enables Rapid and Titratable Conditional Regulation

[0093] To demonstrate the applicability of pRAIDRS as a rescue system in

mammalian stem cells, mouse ESCs ("mESCs") in which the protein level of NANOG is controlled by auxin were engineered. mESCs were infected with pRAIDRS harboring an shRNA targeting the 3 '-untranslated region (3 '-UTR) of Nanog mRNA and an AID ⁴⁷ - fused Nanog coding sequence (A-NANOG) lacking UTRs. As a control, mESCs were infected with pRAIDRS containing only GFP-AID ⁴⁷ (GFP-A). Post-selection clones demonstrated effective silencing of endogenous NANOG by the shRNA, whereas exogenous A-NANOG, which was expressed at levels comparable to endogenous NANOG in control cells, was effectively and rapidly depleted following auxin treatment (FIG 3A and FIG 4A). Phenotypically, auxin treatment of A-NANOG mESCs, but not GFP-A mESCs, resulted in depletion of alkaline phosphatase ("AP") positive colonies, loss of ESC morphology and a transcriptional program characteristic of NANOG inactivation (Ivanova et al., "Dissecting Self-Renewal in Stem Cells with RNA Interference," Nature 442:533-538 (2006), which is hereby incorporated by reference in its entirety), namely downregulation of self-renewal genes and induction of endodermal differentiation markers (FIGs 3B-3D and FIG 4B). A similar transcriptional response was elicited by shRNA-mediated NANOG depletion (FIGs 4C-4D). In contrast, mESCs infected with pRAIDRS harboring a Nanog shRNA and a Nanog coding sequence fused to an irrelevant degron (OsJAZ ³³ , see below) did not respond to auxin treatment (FIGs 4E-4F). These results demonstrate the applicability of pRAIDRS as a molecular switch that facilitates dissection of protein function in mESCs.

[0094] To exemplify the rapidity of degradation enabled by pRAIDRS, a rescue system for the checkpoint kinase CHKl in mESCs was established. CHKl is required for mouse development and its disruption severely impairs DNA damage responses (Liu et al., "Chkl is an Essential Kinase that is Regulated by Atr and Required for the G2/M DNA Damage

Checkpoint," Genes Dev. 14: 1448-1459 (2000) and Takai et al., "Aberrant Cell Cycle

Checkpoint Function and Early Embryonic Death in Chkl(-/-) Mice," Genes Dev. 14: 1439-1447 (2000), which are hereby incorporated by reference in their entirety). Multiple roles are also attributed to CHKl in normal cell cycle progression (Enders GH, "Expanded Roles for Chkl in Genome Maintenance," J. Biol. Chem. 283 : 17749-17752 (2008) and Sorensen et al.,

"Safeguarding Genome Integrity: The Checkpoint Kinases ATR, CHKl and WEE1 Restrain CDK Activity During Normal DNA Replication," Nucleic Acids Res. 40:477-486 (2012), which are hereby incorporated by reference in their entirety) and in mESC self-renewal (Lee et al., "Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling," Cell Stem Cell 11 : 179-194 (2012), which is hereby incorporated by refence in its entirety). mESCs were infected with pRAIDRS harboring a Chkl 30 -UTR-targeting shRNA and an AID47-fused Chkl coding sequence ("A-CHKl"). A western blot analysis of selected clones demonstrated efficient silencing of endogenous CHKl and complete auxin-dependent degradation of A-CHKl (FIG 5A). Next, A-CHKl cells were monitored for the effects of CHKl depletion. When cells were infected and selected in the presence or absence of auxin, a marked auxin-dependent depletion of AP-positive colonies was observed (FIG 6A), apparently supporting the reported roles of CHKl in mESC self-renewal. However, CHKl depletion in post-selection cells had only a marginal effect, if any, on proliferation rate, stage specific embryonic antigen- 1 ("SSEA- 1") levels, mRNA expression patterns or apoptosis (FIGs 6, 7). These data imply that the initial effect of CHK1 depletion may reflect its role during cellular stress responses induced by viral infection or drug selection.

[0095] Next, pRAIDRS was used to study the role of CHK1 in the mESC DNA damage response. To this end, cells were treated with aphidicolin, a DNA polymerase inhibitor that induces DNA breaks and activates the ATR-CHK1 pathway (Feijoo et al., "Activation of Mammalian Chkl During DNA Replication Arrest: A Role for Chkl in the Intra-S Phase Checkpoint Monitoring Replication Origin Firing," J. Cell Biol. 154:913-923 (2001), which is hereby incorporated by reference in its entirety). CHK1 depletion dramatically sensitized mESCs to aphidicolin, as auxin-treated A-CHK1 cells died following treatment with 0.1 mM aphidicolin, whereas control cells survived following treatment with 100-fold higher

concentrations of aphidicolin (FIG 5B). This hypersensitivity was specific to CHK1 depletion as auxinand control -treated GFP-A cells responded indistinguishably to aphidicolin treatment (FIGs 7A-7B). CHK1 depletion in aphidicolin-treated cells resulted in rapid induction of apoptosis, activation of a p53 ("TRP53") transcriptional response, predominantly of the p53 target Fas that encodes a death receptor (Owen-Schaub et al., "Wild-Type Human p53 and a Temperature-Sensitive Mutant Induce Fas/APO-1 Expression," Mol. Cell Biol. 15:3032-3040 (1995), which is hereby incorporated by reference in its entirety), as well as a later induction of differentiation (FIGs 7C-7F). Applicants hypothesize that the aphidicolin susceptibility of CHKl-depleted cells stems from the ability of CHK1 to phosphorylate and induce the cytoplasmic sequestration or degradation of CDC25 phosphatases, which, in turn, augments the inhibitory Tyrl5 phosphorylation of CDK1 ("CDKl ^pY15 "), preventing cell cycle progression (Boutros et al., "CDC25 Phosphatases in Cancer Cells: Key Players? Good Targets?," Nat. Rev. Cancer 7:495-507 (2007), which is hereby incorporated by reference in its entirety). Indeed, rapid (20 minutes) auxin-dependent depletion of CHK1 in aphidicolin-treated mESCs resulted in synchronous mitotic entry 45-90 minutes post-auxin treatment, paralleling CDC25A stabilization and the decrease in CDKl ^pY15 , and preceding p53 stabilization and the induction of Fas mRNA (FIGs 5C-5F and FIG 7G). Thus, depleting CHK1 in DNA-damaged mESCs led to a series of consecutive phenotypes already observable 45 minutes post treatment. Moreover, by titrating down CHK1 levels in DNA-damaged mESCs, the applicability of pRAIDRS as a sensitive analogue tuner that enables fine-tuning of protein levels and their associated phenotypes (FIG 8), facilitating in-depth analyses of protein dose responses was demonstrated.

[0096] Auxin-induced degradation was shown to be reversible (Nishimura et al., "An

Auxin-based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009), which is hereby incorporated by reference in its entirety). To demonstrate this for pRAIDRS, p53-null lung adenocarcinoma cells (NCI-H1299) expressing an auxin-degradable wild-type p53-AID47 (p53-A) were engineered. These cells were infected and cultured in the presence of auxin to prevent the stabilization of p53, known for its ability to inhibit cell growth (Brosh et al., "When Mutants Gain New Powers: News from the Mutant p53 Field," Nat. Rev. Cancer 9:701-713 (2009) and Brosh et al., "Transcriptional Control of the Proliferation Cluster by the Tumor Suppressor p53," Mol. Biosyst. 6: 17-29 (2010), which are hereby incorporated by reference in their entirety). However, following auxin removal p53 was rapidly stabilized, leading to the induction of the p53 target genes p21 (CDKN1 A) and MDM2, and resulting in growth retardation (FIG 9). In sum, these data validate and exemplify pRAIDRS as an easy-to-use single-vector system enabling the construction of highly rapid, titratable, reversible and non-stressful molecular tuners in mESCs and other cell types.

Example 3 - pJAZ Functions as a Coronatine-Induced Degradation Rescue System

[0097] Simultaneous conditional regulation of two proteins represents a powerful tool for complex analyses. Next, a second rescue system that harnesses the plant j asm onate-induced degradation response was engineered. As described above, in plants, isoleucine-conjugated jasmonate ("JA-Ile") mediates the binding of the F-box hormone receptor COI1 and the JAZ degron domain of target proteins, which are consequently ubiquitinated and degraded (Chini et al., "The JAZ Family of Repressors is the Missing Link in Jasmonate Signalling," Nature 448:666-671 (2007) and Owen-Schaub et al., "Wild-Type Human p53 and a Temperature- Sensitive Mutant Induce Fas/ APO-1 Expression," Mol. Cell Biol. 15:3032-3040 (1995), which are hereby incorporated by reference in their entirety) (FIG 1 A). Applicants believe that expression of COI1 in mammalian cells would enable hormone-dependent degradation of JAZ- fused POIs. As mammalian cells lack the pathway for JA-Ile conjugation, coronatine, a bacterial analogue of JA-Ile34 was used. Using the same architecture as pRAIDRS (FIG IB), pJAZ, a vector harboring a codon optimized ^, thaliana COI1 receptor (AtCOIl) and a 23-amino acid JAZ degron ( _^ 4tJAZ23, FIG 10A) that was previously identified as the A. thaliana JAZl minimal degron motif (Sheard et al., "Jasmonate Perception by Inositol-phosphate-potentiated COIl-JAZ Co-Receptor," Nature 468:400-405 (2010), which is hereby incorporated by reference in its entirety) was constructed.

[0098] For initial testing, HEK-293T cells were infected with pJAZ harboring GFP-

AtJAZ ²³ and treated with coronatine. Disappointingly, GFP degradation was extremely ineffective (FIG 11 A, version 1). pJAZ was then systematically and iteratively optimized by testing different C0I1 orthologues and fusion proteins, and by altering the degron length and origin (FIG 11 A and FIGs 10A-10M). It was hypothesized that the lack of coronatine-dependent degradation stems from insufficient binding of^tCOIl to human SKPl (HsSKPl). Accordingly, an OsTIRl ^F"box -,4tCOIl ^LRR chimera composed of OsTHU F-box domain (amino acids 1-39) (Tan et al., "Mechanism of Auxin Perception by the TIR1 Ubiquitin Ligase," Nature 446:640- 645 (2007), which is hereby incorporated by reference in its entirety), which binds HsSKPl effectively (Nishimura et al., "An Auxin-based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009), which is hereby incorporated by reference in its entirety), and AtCOIl leucine-rich repeat (amino acids 52-592), the receptor region responsible for hormone and degron binding (Sheard et al., "Jasmonate Perception by Inositol -phosphate-potentiated COIl-JAZ Co-Receptor," Nature 468:400-405 (2010), which is hereby incorporated by reference in its entirety) was generated. Cells infected with pJAZ version 2 demonstrated -50% coronatine-dependent GFP degradation. A similar chimeric receptor harboring i¾SKP2 ^F"box (version 3) and various ^tCOIl-HsSKPl fusions (versions 4a-d) failed to mediate coronatine-dependent degradation. Next, an extended 31 -amino acid degron (AtJAZ ³¹ ), as well as AtJAZ ^FL , the full-length thaliana JAZ1 protein were tested. Neither ^tJAZ ³¹ nor ^tJAZ ^FL enhanced pJAZ function. To test whether the O^TIRl ^F"box -^tCOIl ^LRR receptor is sufficiently expressed, an amino-terminal haemagglutinin (HA) tag was added. The receptor level was found to be comparable to the level of HA-OsTIRl in pRAIDRS-infected cells (FIG 10E) and, hence, presumably sufficient. Unexpectedly, the HA tag boosted pJAZ efficiency to -70% (version 2 ^HA ), possibly by stabilizing the receptor (Morawska et al., "An Expanded Tool Kit for the Auxin-inducible Degron System in Budding Yeast," Yeast 30:341- 351 (2013), which is hereby incorporated by reference in its entierty). It was next reasoned that at 37°C, a rice coronatine receptor (OsCOIl) might function better than ^tCOIl, as reported for the auxin receptor (Nishimura et al., "An Auxin-based Degron System for the Rapid Depletion of Proteins in Nonplant Cells," Nat. Methods 6:917-922 (2009), which is hereby incorporated by reference in its entierty). Of the three OsCOIl paralogues, OsCOIlB was chosen, as it binds a larger variety of JAZ proteins (Lee et al., "Oryza Sativa COI Homologues Restore Jasmonate Signal Transduction in Arabidopsis coil-1 Mutants," PLoS ONE 8:e52802 (2013), which is hereby incorporated by reference in its entiretly) and tested with either the AtJAZ23 degron or with a 23-amino acid rice degron, OsJAZ ²³ (FIG 10A). Both versions (5- _^ 4t23 and 5-Os23, respectively) were found to be nonfunctional. However, a chimeric receptor (OsTTRl ^F"box - OsCOIlB ^LRR ) comprising OsTIRl F-box domain and OsCOIlB LRR (version 6-Os23) mediated nearly 90% degradation of GFP-Os AZ ²³ . Nevertheless, this version probably suffered from coronatine-independent degradation, as most cells had low fluorescence levels (FIG 10F).

Switching to ^tJAZ ²³ or extending the rice degron to 33 AAs (OsJAZ ³³ ) restored GFP levels, but attenuated the effect of coronatine (versions 6- _^ 4t23 and 6-0^33, respectively). Notably, using AtJAZ ^FL resulted in high GFP expression and 95% coronatine-induced degradation (version 6- AtFV), while conferring nuclear localization to GFP (FIG 10G), in accordance with JAZl localization in plants (Grunewald et al., "Expression of the Arabidopsis Jasmonate Signalling Repressor JAZI/TIFYIOA is Stimulated by Auxin." EMBO Reports 10:923-928 (2009), which is hereby incorporated by reference in its entirety), prompting applicants to believe that its degron efficiency partially derives from its nuclear localization. Accordingly, GFP-OsJAZ ³³ targeted to the nucleus with an NLS (version 7) was found to enhance both dose- and time-dependent coronatine-induced degradation, reaching >95% with 50 mM coronatine (FIG 10H). Thus, a chimeric O5TIRl ^F"box -O5COIlB ^LRR receptor can effectively mediate coronatine-dependent degradation of nuclear POIs fused to an OsJAZ ³³ degron without evidence of coronatine- independent degradation, coronatine receptor-independent degradation, or coronatine toxicity (FIGs 10I-10M). Importantly, pJAZ version 7 (henceforth pJAZ) functioned nearly as well as pRAIDRS in mediating hormone-dependent degradation of nuclear GFP (FIG 1 IB) and, similar to pRAIDRS, pJAZ enabled the engineering of a molecular switch in which an endogenous protein was replaced with a coronatine-regulated exogenous protein, as demonstrated by engineering a p53 switch in human ESCs (/zESCs; FIGs 11C, 1 ID).

[0099] Next, cells expressing coronatine-degradable NLS-GFP-Os AZ ³³ and auxin- degradable NLS-mOrange-AID ⁴⁷ were engineered using pJAZ and pRAIDRS harboring PuroR or BSD, respectively, and selecting these cells with puromycin and blasticidin. Flow cytometric and microscopic analyses demonstrated that pRAIDRS and pJAZ function effectively and independently in a variety of cell types, including hESCs (FIG 1 lE-11G), P19 mouse embryonal carcinoma cells, H1299 lung adenocarcinoma cells, HEK-293T cells, NIH/3T3 mouse embryonic fibroblasts, NCI-H358 human non-small cell lung cancer cells and HCT-116 human colorectal carcinoma cells (FIGs 12A-12G). Importantly, both hormones induced 90-99% degradation, depending on the cell type, and did not show any cross-reactivity or interference, suggesting that neither system saturates the shared ubiquitination machinery. These data validate the applicability of pRAIDRS and pJAZ as a dual analogue molecular tuner.

Example 4 - A Dual Molecular Switch to Dissect the NOTCH1 Pathway

[0100] NOTCH signaling, which is inactive in undifferentiated hESCs, participates in their differentiation into embryonic lineages (Noggle et al., "Notch Signaling is Inactive but Inducible in Human Embryonic Stem Cells," Stem Cells 24: 1646-1653 (2006) and Yu et al., "Notch Signaling Activation in Human Embryonic Stem Cells is Required for Embryonic, but not Trophoblastic, Lineage Commitment," Cell Stem Cell 2:461-471 (2008), which are hereby incorporated by reference in their entirety). In mice, NOTCH was also implicated in

trophectoderm formation (Rayon et al., "Notch and Hippo Converge on Cdx2 to Specify the

Trophectoderm Lineage in the Mouse Blastocyst," Dev. Cell 30:410-422 (2014), which is hereby incorporated by reference in its entirety). Canonical NOTCH signaling involves ligand binding to the membrane receptor, leading to cleavage of the NOTCH intracellular domain ("NICD") and its translocation to the nucleus, where it binds CSL (RBPJ) and MAMLl to activate gene transcription (Andersen et al., "Non-canonical Notch Signaling: Emerging Role and

Mechanism," Trends Cell Biol. 22:257-265 (2012), which is hereby incorporated by reference in its entirety). Applicants sought to construct a molecular switch to dissect NOTCH1 signaling in hESCs. hESCs were infected with pRAIDRS NICD-A, which harbors an shRNA targeting the full-length NOTCH1 receptor and an NICD-AID ⁴⁷ CDS (FIGs 13A-13B). These cells were maintained with auxin to prevent NICD-AID ⁴⁷ accumulation, which occurs quickly following auxin removal (FIG 14A) and induces robust differentiation (FIGs 13C-13D). Next, these cells and their pRAIDRS GFP-A control counterparts were infected with pJAZ harboring a dominant- negative MAMLl (Yu et al., "Notch Signaling Activation in Human Embryonic Stem Cells is Required for Embryonic, but not Trophoblastic, Lineage Commitment," Cell Stem Cell 2:461- 471 (2008), which is hereby incorporated by reference in its entirety) fused to NLS-GFP and OsJAZ ³³ (dnMAMLl-NLS-GFP-OsJAZ ³³ , abbreviated as dnMl-GFP-J), or with pJAZ NLS- GFP-OsJAZ ³³ (GFP-J) as a control. Coronatine treatment effectively induced degradation of dnMl -GFP-J (FIG 14B).

[0101] The effect of NICD-AID ⁴⁷ accumulation following auxin removal in a self- renewal condition was analyzed in the presence of fibroblast growth factor 2 ("FGF2") and transforming growth factor-β (TGFP) or in their absence (differentiation condition). As depicted in FIG 14C and FIG 13E, in pRAIDRS NICD-A hESCs, auxin removal led to the activation of the NOTCH targets HEYl and HES5 in a manner largely independent of FGF2/TGFp.

However, the mesoderm marker T (Brachyury) and the ectoderm marker SOX1 were induced by NICD-A exclusively in the presence of FGF2/TGFP, whereas the endoderm marker GATA6 and the trophectoderm marker GATA3 were induced by NICD-A primarily in the absence of FGF2/TGFP. In nearly all cases, dnMl -GFP-J hindered NICD-A-dependent transactivation and coronatine treatment attenuated the effect of dnMl -GFP-J, restoring gene expression. Moreover, NANOG downregulation following FGF2/TGFP withdrawal was also NICD dependent. Taken together, these data indicate that canonical NOTCH1 signaling can induce key lineage commitment transcription factors in hESCs, and that the identity of these factors depends on FGF2/TGFP, unveiling a cross-talk between NOTCH1 signaling and the self-renewal circuitry. In addition, the induction of GAT A3 implicates NOTCH 1 in hESC trophectodermal

differentiation. These data exemplify the applicability of pRAIDRS and pJAZ for the construction of dual molecular tuners capable of accurate dissection of signaling pathways in hESCs.

Discussion of Examples 1-4

[0102] Examples 1-4 demonstrate a molecular system that facilitates experiments that were previously unfeasible or very complicated in mammalian cells in general and ESCs in particular. Both pRAIDRS and pJAZ are easy-to-construct single vectors (FIG 1 and FIG 16), which deliver all the necessary elements for the construction of rapid and reversible analogue molecular tuners or, when combined, a dual tuner.

[0103] The iterative engineering of pRAIDRS and pJAZ was aimed at enhancing their functionality in ESCs. A 'hormone receptor/P2A/degron-fused POI/P2A/selectable marker' cassette that was codon optimized for human cells is transcriptionally driven by a PGK-1 or EFla promoter. These promoters offer strong and stable expression in a wide variety of cells, with pPGK-1 being more stable in ESCs and pEFla stronger (Norrman et al., "Quantitative Comparison of Constitutive Promoters in Human ES Cells," PLoS ONE 5:el2413 (2010); Xia et al., "Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in a Promoter-dependent Manner," Stem Cells Dev. 16: 167-176 (2007); and Qin et al.,

"Systematic Comparison of Constitutive Promoters and the Doxycycline-inducible Promoter," PLoS ONE 5:el0611 (2010), which are hereby incorporated by reference in their entirety). The P2A peptides separating the aforementioned components are the most effective 2A peptide in mammalian cells (Kim et al., "High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice," PLoS ONE 6:el8556 (2011), which is hereby incorporated by reference in its entirety). The AID degron was minimized fivefold, to reduce interference and spontaneous cleavage. To harness the jasmonate-induced degradation pathway, a chimeric receptor was engineered, as neither thaliana nor rice coronatine receptors function in mammalian cells, and identified the minimal rice JAZ degron motif compatible with this chimeric receptor. The use of selectable markers translated in-frame with the hormone receptor and POI should ensure that drug-resistant cells are hormone sensitive. Finally, the silencing of an endogenous gene-of-interest by the pU6-driven shRNA renders each lentiviral vector an independent rescue system.

[0104] A tetracycline-based complementation approach has proven useful for gene discovery and characterization in ESCs (Lee et al., "Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling," Cell Stem Cell 11 : 179-194 (2012); Ang et al., "Wdr5 Mediates Self-Renewal and Reprogramming Via the Embryonic Stem Cell Core

Transcriptional Network," Cell 145: 183-197 (2011); and Ivanova et al., "Dissecting Self- Renewal in Stem Cells with RNA Interference," Nature 442:533-538 (2006), which are hereby incorporated by reference in their entirety).

Nevertheless, its slowness and the requirement for rtTA expression limit its use. Conversely, pAID enables rapid control of proteins, but does not offer endogenous gene inactivation, uses a large bioactive degron, and is inapplicable to mammalian stem cells (Table 1). Although auxin- dependent degradation was previously harnessed to generate molecular switches in somatic mammalian cells, this was achieved by sequential and laborious steps, such as TIRl

overexpression, POI-degron overexpression and gene-of-interest knockdown/out (Han et al., "Catalytic Assembly of the Mitotic Checkpoint Inhibitor BubRl-Cdc20 by a Mad2-Induced Functional Switch in Cdc20," Mol. Cell 51 :92-104 (2013); Holland et al., "Inducible, Reversible System for the Rapid and Complete Degradation of Proteins in Mammalian Cells," Proc. Natl. Acad. Sci. USA 109:E3350-3357 (2012); and Rodriguez-Bravo et al., "Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad 1 -dependent Anaphase Inhibitor," Cell 156: 1017-1031 (2014), which are hereby incorporated by reference in their entirety) or, alternatively, by genomic targeting of AID degrons to both alleles of the endogenous gene combined with TIRl overexpression (Lambrus et al., "p53 Protects Against Genome Instability Following Centriole Duplication Failure," J. Cell Biol. 210:63-77 (2015), which is hereby incorporated by reference in its entirety). Although these approaches were effective in constructing single molecular tuners, the system of the present invention enables the engineering of a dual molecular tuner with unparalleled simplicity and quickness, and is particularly useful for studying ESCs, which are hard to otherwise manipulate genetically. Importantly, the rapidity of auxin-dependent protein depletion achieved with the pRAIDRS system (20-30 minutes for >95% degradation of NANOG and CHK1) is comparable with those reported by Han et al. (Han et al., "Catalytic Assembly of the Mitotic Checkpoint Inhibitor BubRl-Cdc20 by a Mad2- Induced Functional Switch in Cdc20," Mol. Cell 51 :92-104 (2013), which is hereby incorporated by reference in its entirety) (-90 minutes), Holland et al. (Holland et al., "Inducible, Reversible System for the Rapid and Complete Degradation of Proteins in Mammalian Cells," Proc. Natl. Acad. Sci. USA 109:E3350-3357 (2012), which is hereby incorporated by reference in its entirety) (60-100 minutes), Rodriguez-Bravo et al. (Rodriguez-Bravo et al., "Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad 1 -dependent Anaphase Inhibitor," Cell 156: 1017-1031 (2014), which is hereby incorporated by reference in its entirety) (>120 min) and Lambrus et al. (Lambrus et al., "p53 Protects Against Genome Instability Following

Centriole Duplication Failure," J. Cell Biol. 210:63-77 (2015), which is hereby incorporated by reference in its entirety) (10-30 minutes) in mammalian cells.

[0105] pRAIDRS and pJAZ combine the advantages of the genetic complementation and hormone-induced degradation strategies, while averting their limitations, as each vector represents a fully functional rescue system specifically tailored to mammalian stem cells and both offer rapid, reversible and titratable control of protein levels. Importantly, combining endogenous gene silencing with conditional rescue ensures high-confidence genotype-to- phenotype causal linkages. Moreover, in contrast to other conditional protein

degradation/activation systems (Bonger et al., "Small-Molecule Displacement of a Cryptic Degron Causes Conditional Protein Degradation," Nat. Chem. Biol. 7:531-537 (2011);

Banaszynski et al., "A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules," Cell 126:995-1004 (2006); Neklesa et al., "Small-Molecule Hydrophobic Tagging-Induced Degradation of HaloTag Fusion Proteins," Nat. Chem. Biol. 7:538-543 (2011); and Braselmann et al., "A Selective Transcriptional Induction System for Mammalian Cells Based on Gal4-estrogen Receptor Fusion Proteins," Proc. Natl. Acad. Sci. USA 90: 1657-1661 (1993), which are hereby incorporated by reference in their entirety), pRAIDRS and pJAZ degrons are extremely short, diminishing interference with POI localization and function. Other advantages of pRAIDRS and pJAZ are listed in Table 1. Of note, although both pRAIDRS and pJAZ enable hormone-dependent degradation of cytoplasmic and nuclear POIs, with both systems the degradation of nuclear POIs is faster and requires lower hormone concentrations.

[0106] As a proof-of-concept, a molecular switch for the ESC master regulator NANOG was constructed. This switch enabled conditional and nearly complete rapid depletion of NANOG, recapitulating its well-established roles in mESCs (Cavaleri et al., "Nanog: A New Recruit to the Embryonic Stem Cell Orchestra," Cell 113 :551-552 (2003), which is hereby incorporated by reference in its entirety). By engineering a molecular switch for CHK1, a series of gene-specific phenotypes were elicited as early as 45 minutes following hormone treatment. This degree of rapidity can facilitate the distinction between primary and secondary events, and enables high-resolution kinetic studies. Furthermore, owing to the inert and specific nature of hormone-induced degradation, only minor effects were observed following CHK1 depletion in post-selection cells, contrasting with the current conception of the role of CHK1 in normal cycling cells (Enders GH, "Expanded Roles for Chkl in Genome Maintenance," J. Biol. Chem. 283 : 17749-17752 (2008); Sorensen et al., "Safeguarding Genome Integrity: The Checkpoint Kinases ATR, CHK1 and WEE1 Restrain CDK Activity During Normal DNA Replication," Nucleic Acids Res. 40:477-486 (2012); and Niida et al., "Depletion of Chkl Leads to Premature Activation of Cdc2-cyclin B and Mitotic Catastrophe," J. Biol. Chem. 280:39246-39252 (2005), which are hereby incorporated by reference in their entirety) and in mESC self-renewal (Lee et al., "Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling," Cell Stem Cell 11 : 179-194 (2012), which is hereby incorporated by reference in its entirety).

Conversely, it was demonstrated that CHK1 plays a crucial protective role in DNA-damaged mESCs by restricting mitotic entry, which otherwise leads to apoptosis or differentiation. The CHK1 molecular switch represents a unique tool for screening and characterizing CHK1 inhibitors and DNA-damage sensitizers, a rapidly growing category of anti-cancer drugs (Zhang et al., "Roles of Chkl in Cell Biology and Cancer Therapy," Int. J. Cancer 134: 1013-1023

(2014) and Ma et al., "Death by Releasing the Breaks: CHK1 Inhibitors as Cancer Therapeutics," Trends Mol. Med. 17:88-96 (2011), which are hereby incorporated by reference in their entirety).

[0107] Applicants also engineered cancer cells expressing hormone-degradable p53 and demonstrated its unleashing by auxin removal (Brosh et al., "Transcriptional Control of the Proliferation Cluster by the Tumor Suppressor p53," Mol. Biosyst. 6: 17-29 (2010), which is hereby incorporated by reference in its entirety), highlighting the rapid reversibility of hormone- induced degradation. Stable ectopic expression of tumour suppressors is cumbersome, as cancer cells quickly evade their effects. However, effective auxin-induced p53 degradation enabled prolonged propagation of these cells without growth inhibition or transgene silencing.

Applicants further demonstrated how pRAIDRS and pJAZ allow titratable control of protein levels, a feature that enables studies of protein dose responses and threshold levels.

[0108] By engineering a coronatine-dependent p53 switch, the applicability of pJAZ for rapid and simple construction of molecular switches in hESCs was demonstrated. Moreover, applicants showed how combining pRAIDRS and pJAZ yields a dual molecular switch, where auxin and coronatine control two different proteins independently. Applying this method to hESCs, unknown aspects of the canonical NOTCHl pathway and its integration with the hESC self-renewal network were unveiled. Thus, the generation of such dual switches (or tuners) is valuable for dissecting the function of proteins and regulatory networks. [0109] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.