System and method for checking the ability of a protein of interest to act as a substrate for an enzyme

Abstract

The invention concerns a system for checking whether a protein of interest (POI) can act as a substrate, in particular a favorable substrate, for an enzyme selected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase in a eukaryotic cellular environment, wherein the system comprises a first recombinant protein comprising an amino acid sequence of the selected enzyme and an amino acid sequence of a first binding protein of a dimerization system, a second recombinant protein comprising an amino acid sequence of the POI, an amino acid sequence of a reporter protein and an amino acid sequence of a second binding protein of the dimerization system and a ligand of the dimerization system.

Description

[0001]System and method for checking the ability of a protein of interest to act as a sub- strate for an enzyme The present invention concerns a system and a method for checking whether a protein of interest (POI) can act as a substrate for an enzyme and a use of such a system. The enzyme may in particular be an E3-ubiquitin ligase. According to Varnai P. et al., "Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells" J Cell Biol.2006 Nov 6;175(3), pages 377-382 rapamycin (rapa)-in- duced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) en- zyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinosi- tol 4,5-bisphosphate (PtdIns(4,5)P2) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P2 as monitored by the PLCδ1PH-GFP fusion construct. It was demonstrated that rapid inducible depletion of PM PtdIns(4,5)P2 is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P2 depletion. PM PtdIns(4,5)P2 is a phospholipid of the plasmamembrane. The document does not concern degradation of a protein as a substrate for an enzyme. From WO 2021 / 180787 A1 a Proteolysis Targeting Chimera (PROTAC) for degra- dation of Aurora A-kinase is known. The PROTAC has the chemical structure AAB-L-E3B, wherein AAB is a binding unit for Aurora A-kinase, L is a connecting unit and E3B is a binding unit for E3-ubiquitin ligase Cereblon. It has been recognized that not all PROTACs are able to combine a POI that shall be degraded with an E3-ubiquitin ligase such that the POI is degraded or effec- tively degraded in a eukaryotic cellular system as a consequence of ubiquitination by the E3-ubiquitin ligase. This may be because the specific POI cannot act as a 636399-Uni Würzburg-an-1 substrate or at least not a favorable substrate for the E3-ubiquitin ligase and the degradation process following ubiquitination by the E3-ubiquitin ligase. The main reasons for this may be the expression of E3-ubiquitin ligase and POI in the differ- ent cellular compartments, incompatibility of the POI / E3-ubiquitin ligase to form ternary complex, absence of lysine residue on the POI surface in the vicinity of the E3-ubiquitin ligase machinery, and absence of polyubiquitin chain linkage (like ly- sine-48 linked ubiquitin chain) activity of the E3-ubiquitin ligase machinery that tar- gets the ubiquitinated POI to 26S proteasome for degradation. Since the effort for synthesizing a PROTAC is relatively high, the inventors of the present invention recognized that there is a general need for a screening system for testing whether a specific protein of interest can act as a substrate, in particular a favorable sub- strate, for a specific enzyme. From Qian, Shu-Bing et al., J. Biol. Chem.284, 2009, pages 26797 to 26802, the use of an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase (CHIP) as a model E3 system to investigate the role of substrate positioning in its ubiquitination is known. One domain of this CHIP was replaced with FK506 binding protein (FKBP) without resulting in a loss of its enzymatic ac- tivity. FKBP and FKBP12-rapamycin binding domain (FRB) dimerize with high af- finity in the presence of rapamycin. This property was used to test whether the lig- ase activity of the CHIP-FKBP construct can be redirected toward new substrates. Therefore, a FRB-fused green fluorescence protein (GFP) was constructed. All protein constructs were expressed in Escherichia coli, purified and applied for ubiquitination in an in vitro assay. The authors found that addition of rapamycin to the FRB-GFP and the FKBP-CHIP resulted in efficient ubiquitin conjugation on FRB-GFP. Deng, W. et al. "Tunable light and drug induced depletion of target proteins", Nat Commun 11, 304 (2020) discloses a system for a rapamycin-induced depletion of a GFP-POI fusion protein. In this system a destruction module (E3, ubiquitin E3 ligase) and a targeting module (Nb, Nanobody) are fused to FRB and FKBP, re- spectively. Upon binding of the GFP-POI fusion protein to the nanobody and 636399-Uni Würzburg-an-1 addition of rapamycin FRB and FKBP heterodimerized and brought the GFP-POI fusion protein and the destruction module into close proximity. This caused the GFP-POI fusion protein to be ubiquitinated and thus marked for proteasomal deg- radation. Kanner S. A. et al. "Sculpting ion channel functional expression with engineered ubiquitin ligases", eLife 2017; 6:e29744 discloses fusion proteins comprising the catalytic domain of E3 ligase, CHIP, fused to FRB, a YFP-binding (nano) domain fused to FKBP and a voltage-gated K+channel pore-forming subunit, Q1, fused to YFP. Upon addition of rapamycin FKBP / FRB dimerized and brought the nano- bound Q1-YFP in proximity to FRB-CHIP which resulted in ubiquitination and deg- radation of Q1-YFP. Lazarou, M. et al., Developmental Cell 22, 2012, pages 320 to 333, discloses an assay in which the FKBP domain was fused to mCherry-Parkin, wherein Parkin is a cytosolic E3 ligase. Furthermore, the mitochondrial protein Fis1 was tagged with FRB. Upon treatment of cells containing both constructs with a rapalog, mCherry- FKBP-Parkin was recruited to mitochondria. However, the mCherry-FKBP-Parkin targeted to mitochondria by FRB-Fis1 did not ubiquitinate mitochondria. From Pinch, B. J. et al., "A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes", bioRxiv, 2020 (https: / / doi.org / 10.1101 / 2020.08.13.249482) a method for evaluating the ability of recombinant E3 ligase components to support neo-substrate degradation is known. The method makes use of Covalent Functionalization Followed by E3 Electroporation (COFFEE) into live cells. For example a recombinant von Hippel- Lindau (VHL) E3 ligase was functionalized via its solvent-exposed cysteines using a simple maleimide warhead linked to the BRD4 ligand JQ1. This functionalized recombinant E3 ligase was then electroporated into live cells to form functional E3- ubiquitin ligase complexes capable of catalyzing degradation of the target protein BRD4. Degradation of BRD4 was assessed by immunoblot analysis. 636399-Uni Würzburg-an-1 Diehl, C. J. and Ciulli, A. "Discovery of small molecule ligands for the von Hippel- Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders", Chem. Soc. Rev., 2022, 51, 8216 is a review of VHL ligands and PROTAC de- graders. PROTACs are bifunctional molecules designed to simultaneously bind to an E3 ligase and a target protein to induce target ubiquitination and degradation. The document discloses various target proteins, VHL and PROTACs. The purpose of the present invention is to provide an alternative system and method as well as a use of the system for checking whether a specific protein of interest (POI) can act as a substrate for a specific enzyme selected from an E3- ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase. The problem of the invention is solved by present claims 1, 11 and 13. Embodi- ments of the invention are subject-matter of claims 2 to 10, 12 and 14 to 17. According to the invention a system for checking whether a protein of interest (POI) can act as a substrate, in particular a favorable substrate, for an enzyme se- lected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase in a eukaryotic cellular environment is provided. The system comprises - a first recombinant protein comprising or consisting of - an amino acid sequence of the enzyme and an amino acid se- quence of a first binding protein of a dimerization system dimerizing the first binding protein with a second binding protein upon addition of a ligand, wherein a first linker is optionally arranged between the amino acid sequence of the enzyme and the amino acid sequence of the first binding protein or 636399-Uni Würzburg-an-1 - a first recombinant nucleic acid coding for the first recombinant protein, - a second recombinant protein comprising or consisting of - an amino acid sequence of the POI and an amino acid sequence of the second binding protein of the dimerization system, wherein a sec- ond linker is optionally arranged between the amino acid sequence of the POI and the amino acid sequence of the second binding protein, and - an amino acid sequence of a reporter protein or reporter peptide, wherein a third linker is optionally arranged between the amino acid sequence of the POI and the amino acid sequence of the reporter protein or reporter peptide or between the amino acid sequence of the second binding protein and the amino acid sequence of the re- porter protein or reporter peptide, or - a second recombinant nucleic acid coding for the second recombinant pro- tein and - the ligand. The invention makes it possible to analyze for any protein that is of interest (POI) whether it can act as a substrate for any enzyme selected from an E3-ubiquitin lig- ase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase. In case the POI can act as a substrate for one of the mentioned enzymes, the POI is ubiquitinated in case the enzyme is an E3-ubiquitin ligase, SUMOylated in case the enzyme is a SUMO E3 ligase, deubiquitinated in case the enzyme is a deubiq- uitinating enzyme, phosphorylated, in case the enzyme is a kinase or dephosphor- ylated in case the enzyme in a phosphatase. As a consequence, the POI is 636399-Uni Würzburg-an-1 degraded, e. g. in case the POI is ubiquitinated, or stabilized, e. g. in case the POI is deubiquitinated, in the eukaryotic cellular environment. In case the POI is SUMOylated the SUMOylation may either stabilize the POI by preventing ubiquiti- nation and proteasomal degradation or destabilized by mediating UPS mediated degradation. In any case the enzymatic activity can change the cellular concentra- tion of the POI. The eukaryotic cellular environment may be provided in a mamma- lian cell, a fungal cell, in particular a yeast cell, or a plant cell. All eukaryotic cells comprise an E3 machinery and proteasome, i. e. an Ubiquitin Proteasome System (UPS). The eukaryotic cellular environment may be any environment comprising an E3 machinery and proteasome, i. e. the Ubiquitin Proteasome System (UPS). The dimerization system is a so called chemically induced dimerization system. It may comprise or a consist of the first binding protein, the second binding protein and the ligand. The first binding protein and the second binding protein may be identical or different. In case the first binding protein and the second binding pro- tein are different, the dimerization system is a heterodimerization system. In an embodiment of the invention the dimerization system is a heterodimerization sys- tem which is schematically shown in Fig.1, wherein the ligand is designated as "Dimerizer". Any of the first linker, the second linker and the third linker may independently from each other consist of at least one and at most 75, in particular at most 40, in particular at most 20, amino acid residues and / or consist of glycine and / or serine residue(s). The glycine (G) and serine (S) residues may be present in the linker in any combination. Alternatively, any of the first linker, the second linker and the third linker may independently from each other comprise or consist of any of the sequences SEQ ID NO 3 to SEQ ID NO 5 and SEQ ID NO 34 to SEQ ID NO 36 or an oligomer, in particular a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer or nonamer, of any of these sequences, in particular of any of sequences SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, in particular of sequence SEQ ID NO 3 in particular a dimer of SEQ ID NO 3 which is identical with SEQ ID NO 4. 636399-Uni Würzburg-an-1 Nucleotide sequences SEQ ID NO 6 to 8 depict the GSSGGSSG linker (SEQ ID NO 4) along with the restriction site used for cloning. SEQ ID NO 3 to SEQ ID NO 8 and SEQ ID NO 34 to SEQ ID NO 36 are as fol- lows: SEQ ID NO 3: GSSG SEQ ID NO 4: GSSGGSSG SEQ ID NO 5: GGGS SEQ ID NO 6: GAATTCGGGAGCTCCGGTGGGAGCTCCGGT SEQ ID NO 7: ACGCGTGGGAGCTCCGGTGGGAGCTCCGGT SEQ ID NO 8: GGGAGCTCCGGTGGGAGCTCCGGTACGCGT SEQ ID NO 34: GGGGS SEQ ID NO 35: GGSG SEQ ID NO 36: GSGS The principle of the present invention is that the system comprises components that allow to bring the POI and the enzyme in proximity to each other in a eukary- otic cellular environment by dimerization of the first binding protein part of the first recombinant protein and the second binding protein part of the second recombi- nant protein by use of the ligand. For this purpose the first recombinant protein and the second recombinant protein can be expressed in a eukaryotic cell and the ligand can be introduced into the cells e. g. by use of electroporation and / or a sub- stance promoting cellular uptake such as dimethyl sulfoxide (DMSO). The principle of the present invention is schematically shown in Fig.2 except that the reporter protein or reporter peptide at the POI is missing. In Fig.2 the POI is designated as "target", the enzyme E3-ubiquitin ligase is designated as "E3 ligase", the first bind- ing protein is FRB in the left part of Fig.2 and FKBP12 in the right part of Fig.2, the second binding protein is FKBP12 in the left part of Fig.2 and FRB in the right part of Fig.2 and the ligand is rapamycin. As can be seen from a comparison of the left part and the right part of Fig.2, it is irrelevant whether the amino acid se- quence of the first binding protein in the first recombinant protein comprising the 636399-Uni Würzburg-an-1 enzyme and the amino acid sequence of the second binding protein in the second recombinant protein comprising the POI (target) is that of FRB or that of FKBP12. It is only important that the first binding protein and the second binding protein are chosen such that the first binding protein can bind to the second binding protein via the ligand. The change in POI concentration can be detected by use of the reporter protein or reporter peptide. The reporter protein or reporter peptide is any protein or peptide that can be detected directly or indirectly, e. g. after conversion of a further sub- strate by the reporter protein or reporter peptide, by physical means, in particular by detection of luminescence, in particular fluorescence or chemiluminescence. In this way suitable combinations of POIs and enzymes in which the POIs can act as substrates for the enzymes can be found with very limited effort. It is in particular not required to perform chemical synthesis for finding suitable combinations of POIs and enzymes as it is the case when a Proteolytic Targeting Chimera (PROTAC) is generated for this purpose. The first recombinant protein and the second recombinant protein can be generated by genetic engineering. Further- more, compared to results obtainable by use of a PROTAC the use of the system according to the invention can provide results in shorter time with less costs. Due to the reporter protein or reporter peptide the readout of the results is simple and enables a use of the system according to the invention in high-throughput screen- ing. The system and its use are well suited for commercial use. The system may be provided as a kit for checking whether a protein of POI can act as a substrate, in particular a favorable substrate, for an enzyme selected from an E3-ubiquitin lig- ase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase in a eukaryotic cellular environment, wherein the kit comprises all components of the system according to the invention. However, it is also possible to provide the sys- tem according to the invention by separately procuring and then combining the in- dividual components of the system. It is also possible to provide the system ac- cording to the invention by providing a kit comprising a part of the components of the system and to procure the part(s) of the system not comprised by the kit 636399-Uni Würzburg-an-1 separately and then combining the kit with the part(s) of the system not comprised by the kit. In case the enzyme is E3-ubiquitin ligase, the synthesis of a successful PROTAC is very promising for the combinations of POI and enzyme for which it has been found by use of the system according to the invention that the POI can act as a substrate for the enzyme. Since the effort for synthesizing a PROTAC is relatively high and many PROTACs do not work because the POI cannot act as a substrate for a specific enzyme, the system according to the invention reduces the effort for finding successful PROTACs. The first binding protein of the dimerization system may be FKBP-rapamycin bind- ing domain of mTOR complex 1 (FRB) or its analog FRBT78Lor another analog of FRB and the second binding protein of the dimerization system may be 12-kDa FK506 binding protein (FKBP12) or an analog thereof. Alternatively, the first binding protein of the dimerization system may be 12-kDa FK506 binding protein (FKBP12) or an analog thereof and the second binding pro- tein of the dimerization system may be FKBP-rapamycin binding domain of mTOR complex 1 (FRB) or its analog FRBT78Lor another analog of FRB. In both of the above cases the ligand may be rapamycin or an analog of rapamy- cin which analog is able to dimerize FRB or FRBT78Land FKBP12. In particular the analog may be AP21967 in case that the first binding protein or the second binding protein is FRBT78L. Alternatively, the analog may be temsirolimus, everolimus, ridaforolimus, umirolimus, or zotarolimus. However, in principle any chemically in- duced dimerization system can be used. Any of the before mentioned analogs of FRB and FKBP12 consists of an amino acid sequence having a sequence identity of at least 93 % or a similarity of at least 90 % to the amino acid sequence of FRB and FKBP12, respectively. The amino 636399-Uni Würzburg-an-1 acid sequence of FRB may be the amino acid sequence corresponding to nucleic acid sequence SEQ ID NO 11, i. e. SEQ ID NO 33: MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQ AYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK The amino acid sequence of FKBP12 may be amino acid sequence SEQ ID NO 13. The sequence similarity is calculated according to the EMBOSS needle algo- rithm having an open gap penalty of 10.0 and an extended gap penalty of 0.5 and using the Blosum 62 matrix. Sequence similarity in the context of the present invention is determined according to the EMBOSS needle algorithm. The EMBOSS needle algorithm is the standard algorithm for aligning a first amino acid sequence, in particular a first protein, a first polypeptide residue or a first oligopeptide residue, and a second amino acid se- quence, in particular a second protein, a second polypeptide residue or a second oligopeptide residue, over the whole length of both the first amino acid sequence and the second amino acid sequence. The EMBOSS needle algorithm implements the Needleman-Wunsch algorithm (Needleman S.B. and Wunsch C.D., 1970 J. Mol. Biol.48, 443 – 453), wherein a penalty for a gap of n positions is computed according to the following formula: open gap penalty + n – 1 x extended gap pen- alty. The entire length of the first amino acid sequence and the second amino acid se- quence is aligned, and there is no penalty for hanging ends of the overlap. A “gap” designates one or more amino acid residue(s) not being identical in the aligned sequences. The open gap penalty in the context of the present invention is 10.0. The extended gap penalty in the context of the present invention is 0.5. The scoring matrix for comparing amino acid similarities in the context of the present invention is the Blosum 62 matrix. The open gap penalty, the extended gap penalty and the Blosum 62 matrix are standard parameters used in the art. In 636399-Uni Würzburg-an-1 pairwise sequence alignment, the open gap penalty refers to the penalty for opening a gap in the alignment. The open gap penalty does not penalize terminal gaps. In pairwise sequence alignment, the extended gap penalty refers to the penalty for extending a gap by one residue. The extended gap penalty does not penalize terminal gaps. Sequence alignments can be performed with these parameters, e.g. by use of publicly available free tools. For example, EMBL-EBI offers in its assortment of internet tools, e. g. under https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / , a free pairwise sequence alignment service in which the parameters of the present invention can be chosen. Examples of suitable dimerization systems are as follows: 1) GAI (gibberellin insensitive) and GID1 (gibberellin insensitive dwarf 1) system: a) GAI-tag sequence (91 amino acids): SEQ ID NO 16 b) GID1-tag sequence (344 amino acids): SEQ ID NO 17 c) Dimerization agent: (1) Gibberellic acid: IUPAC: (1S,2S,4aR,4bR,7S,9aS,10S,10aR)-2,7-dihydroxy-1-methyl-8-meth- ylene-13-oxo-1,2,4b,5,6,7,8,9,10,10a-decahydro-4a,1-(epoxymeth- ano)-7,9a-methanobenzo[a]azulene-10-carboxylic acid or (2) Gibberellic acid acetoxymethyl ester: IUPAC: 636399-Uni Würzburg-an-1 acetoxymethyl (1S,2S,4aR,4bR,7S,9aS,10S,10aR)-2,7-dihydroxy-1- methyl-8-methylene-13-oxo-1,2,4b,5,6,7,8,9,10,10a-decahydro-4a,1- (epoxymethano)-7,9a-methanobenzo[a]azulene-10-carboxylate 2) Halo-tag (engineered Haloalkane dehalogenase) and SNAP-Tag (Mutants of the DNA repair protein O6-alkylguanine-DNAalkyltransferase) a) Halo-tag sequence (296 amino acids): SEQ ID NO 18 b) SNAP-Tag sequence (181 amino acids): SEQ ID NO 19 c) Dimerization agent: HAXS8: IUPAC: N-[[4-[[(2-Amino-9-H-purin-6-yl)oxy]methyl]phenyl]methyl]-2-[4-[(18- chloro-3,6,9,12-tetraoxaoctadec-1yl)oxy]-2,3,5,6-tetrafluorophen- oxy]acetamide 3) ABI (Abscisic Acid insensitive) and PYL1 (Pyrabactin resistance like) a) ABI-tag sequence (298 amino acids): SEQ ID NO 20 b) PYL1-tag sequence (176 amino acids): SEQ ID NO 21 c) Dimerizing agent: Abscisic acid: IUPAC: (2Z,4E)-5-[(1S)-1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-en-1-yl]- 3-methylpenta-2,4-dienoic acid 4) ABI (Abscisic Acid insensitive) and PYR1 (Pyrabactin resistance 1) a) ABI-tag sequence (298 amino acids): SEQ ID NO 20 636399-Uni Würzburg-an-1 b) PYR1-tag sequence (190 amino acids): SEQ ID NO 22 c) Dimerizing agent: Mandipropamid: IUPAC: 2-(4-chlorophenyl)-N-[2-(3-methoxy-4-prop-2-ynoxyphenyl)ethyl]-2- prop-2-ynoxyacetamide 5) DHFR (E. coli dihydrofolate reductase) and Halo-tag (engineered Haloalkane dehalogenase) a) DHFR-tag sequence (158 amino acids): SEQ ID NO 23 b) Halo-tag sequence (296 amino acids): SEQ ID NO 18 c) Dimerizing agent: Trimethoprim-HaloTag ligand (TMP-Htag): IUPAC: N1-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-N4-(3-(4-((2,4-diaminopy- rimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)propyl)succinamide In case any of these dimerization systems is part of the system according to the in- vention the first binding protein of the dimerization system may be GAI-tag sequence SEQ ID NO 16 and the second binding protein of the dimerization system may be GID1- tag sequence SEQ ID NO 17 and the ligand may be gibberellic acid or gibberellic acid acetoxymethyl ester, or 636399-Uni Würzburg-an-1 the first binding protein of the dimerization system may be GID1-tag sequence SEQ ID NO 17 and the second binding protein of the dimerization system may be GAI-tag sequence SEQ ID NO 16 and the ligand may be gibberellic acid or gibber- ellic acid acetoxymethyl ester, or the first binding protein of the dimerization system may be Halo-tag sequence SEQ ID NO 18 and the second binding protein of the dimerization system may be SNAP-Tag sequence SEQ ID NO 19 and the ligand may be HAXS8, or the first binding protein of the dimerization system may be SNAP-Tag sequence SEQ ID NO 19 and the second binding protein of the dimerization system may be Halo-tag sequence SEQ ID NO 18 and the ligand may be HAXS8, or the first binding protein of the dimerization system may be ABI-tag sequence SEQ ID NO 20 and the second binding protein of the dimerization system may be PYL1-tag sequence SEQ ID NO 21 and the ligand may be abscisic acid, or the first binding protein of the dimerization system may be PYL1-tag sequence SEQ ID NO 21 and the second binding protein of the dimerization system may be ABI-tag sequence SEQ ID NO 20 and the ligand may be abscisic acid, or 636399-Uni Würzburg-an-1 the first binding protein of the dimerization system may be ABI-tag sequence SEQ ID NO 20 and the second binding protein of the dimerization system may be PYR1-tag sequence SEQ ID NO 22 and the ligand may be mandipropamid, or the first binding protein of the dimerization system may be PYR1-tag sequence SEQ ID NO 22 and the second binding protein of the dimerization system may be ABI-tag sequence SEQ ID NO 20 and the ligand may be mandipropamid, or the first binding protein of the dimerization system may be DHFR-tag sequence SEQ ID NO 23 and the second binding protein of the dimerization system may be Halo-tag sequence SEQ ID NO 18 and the ligand may be trimethoprim-HaloTag ligand (TMP-Htag), or the first binding protein of the dimerization system may be Halo-tag sequence SEQ ID NO 18 and the second binding protein of the dimerization system may be DHFR-tag sequence SEQ ID NO 23 and the ligand may be trimethoprim-HaloTag ligand (TMP-Htag). In case the eukaryotic cellular environment is provided in a mammalian cell, a fun- gal cell or a plant cell it is possible to determine whether the POI is a real substrate in the specific cell of the eukaryotic cellular environment. In contrast to an extracel- lular artificial system, this allows a more reliable prediction as to whether the POI is a substrate for the selected enzyme in a real biological system. In the system known from Deng, W. et al., Nat Commun 11, 304 (2020), the condi- tions must always be such that they allow the nanobody to bind to the GFP-POI 636399-Uni Würzburg-an-1 fusion protein. If the extend of GFP-POI fusion protein degradation is low, the rea- son for that can always be poor binding of the nanobody to the GFP-POI fusion protein. Thus, the result does not reliably reflect the degradability of the POI. Simi- larly, in the system known from Kanner S: A: et al., eLife 2017;6:e29744 the condi- tions must always be such that they allow nano to bind to Q1-YFP. If the extend of Q1-YFP degradation is low, the reason for that can be poor binding of nano to Q1- YFP. Thus, the result does not reliably reflect the degradability of Q1 which is the POI in this case. A reason for poor binding in these systems can be, for example, a condition within the cell in which the binding shall take place, or a spatial hinderance of the binding caused by the POI or Q1. The system according to the present invention is not susceptible to this type of error and therefore provides more reliable results. Furthermore, there is the possibility in the systems known from Deng, W. et al. and Kanner S. A. et al. that the tags GFP and YFP rather than the POI and Q1 are ubiquitinated giving rise to false positive results. This possibility is excluded in the system and the method according to the invention by the absence of lysine resi- dues in the amino acid sequences of the reporter protein or reporter peptide, the second binding protein and the second linker and / or third linker. The sequence identity may be at least 95 %, in particular at least 96 %, in particu- lar at least 97 %, in particular at least 98 %, in particular at least 99 % and at most less than 100 % , in particular at most 99.99 %. Sequence similarity may be at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 % and at most less than 100 % , in particular at most 99.99 %. Many of the enzymes selected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase act on lysine residues of their substrate. In case the enzyme acts on lysine residue(s) of the POI it may be that it also acts on lysine residue(s) of the second binding protein and / or lysine residues of the reporter protein or reporter peptide and / or of the second linker and / or of the 636399-Uni Würzburg-an-1 third linker in case the second linker and / or the third linker is / are present in the second recombinant protein. For example, this may be the case when the enzyme is E3-ubiquitin ligase, the reporter protein is NanoLuc® and the second binding protein is FKBP12. In case of ubiquitination of FKBP12 and / or NanoLuc® followed by degradation of the second recombinant protein it would not be possible to de- termine whether degradation was caused by ubiquitination of the POI, of FKBP12 or of NanoLuc®. For restricting the potential activity of the enzyme to the POI, the second recombinant protein may be constructed such that in the second recombi- nant protein - the reporter protein or reporter peptide and the second binding protein and the second linker and / or third linker in case of presence in the second re- combinant protein are chosen such that no lysine residue is present in the amino acid sequences of the reporter protein or reporter peptide, the sec- ond binding protein and the second linker and / or third linker or - any lysine residue in the amino acid sequence of the reporter protein or re- porter peptide, in the amino acid sequence of the second binding protein, in particular of FKBP12 or the analog thereof or of FRB or of its analog FRBT78Lor of the other analog of FRB, and in the amino acid sequence of the second linker and / or third linker in case of presence in the second re- combinant protein is replaced by an arginine residue or histidine residue, in particular an arginine residue. This feature prevents false positive results obtained by enzymatic conversion, in particular ubiquitination, of other parts of the second recombinant protein than the POI. Compared to other systems known in the art this makes the system accord- ing to the invention much more reliable for checking whether a POI can act as a substrate for the selected enzyme. 636399-Uni Würzburg-an-1 In an embodiment of the invention the system comprises the first recombinant pro- tein and the second recombinant protein and the ligand. In another embodiment of the invention the system comprises the first recombinant nucleic acid and the sec- ond recombinant nucleic acid and the ligand. The first recombinant nucleic acid may be contained in an expression vector, in particular a plasmid or a virus or a lentiviral vector. The second recombinant nucleic acid may be contained in the same or a further expression vector, in particular the plasmid or a further plasmid or the virus or a further virus or the lentiviral vector or a further lentiviral vector. The expression vector and the further expression vector may be in each case in- dependently from each other a plasmid or a virus. Alternatively, the first and the second recombinant nucleic acid may be contained in the genome of an animal cell, in particular a mammalian cell, after introduction by CRISPR knock-in tech- nique. In particular the amino acid sequence of an E3-ubiquitin ligase can be cou- pled, i. e. tagged, with the amino acid sequences of the first binding protein and / or the amino acid sequence of an oncogenic protein, such as MYC, WDR5 and AURKA, can be coupled, i. e. tagged, with the amino acid sequences of the sec- ond binding protein and of the reporter protein or reporter peptide in their endoge- nous genomic locus using CRISPR knock-in. In case of envisaged tumor treat- ment, the E3-ubiquitin ligase can be an E3-ubiquitin ligase that is tissue specific and tissue essential. The reporter protein or reporter peptide may be a luciferase, in particular firefly-lu- ciferase, 19 kDa catalytic subunit of Oplophorus luciferase, in particular a lucifer- ase having sequence SEQ ID NO 1 (NanoLuc®), or a luciferase having sequence SEQ ID NO 2 (HiBiT), or a fluorophore, in particular green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein(CFP), blue fluores- cent protein (BFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), far-red fluorescent protein, infra-red fluorescent protein, mNeonGFP (mNG), enhanced GFP (EGFP) or mCherry. EGFP is a basic green fluorescent protein derived from Aequorea Victoria. 636399-Uni Würzburg-an-1 The E3-ubiquitin ligase may be von Hippel-Lindau E3-ubiquitin ligase (VHL), F-box protein (FBXL12) of SCF (Skp1-CUL-1-F-box protein) type E3 ubiquitin ligase complex or Cereblon E3-ubiquitin ligase (CRBN). The POI may be an oncoprotein, i. e., a protein promoting cancer growth or having a gain of function mutation in a cancer cell. The oncoprotein may be oncoprotein Myc, oncoprotein MycN , onco- protein MycL, aurora kinase A (AURKA), aurora kinase B (AURKB), BCR-ABL fu- sion protein, signal transducer and activator of transcription 3 (STAT3), GTPase Kras (KRAS), EWS–FLI fusion protein, epidermal growth factor receptor (EGFR), Erb-b2 receptor tyrosine kinase 2 (ERBB2), ALK tyrosine kinase receptor (ALK), Wee1-like protein kinase (WEE1), catenin beta-1 (CTNNB1) or Yes-associated protein 1 (YAP1). The POI may also be a cofactor of an oncoprotein, i. e., a protein promoting the oncogenicity of an oncoprotein. The cofactor may be WD repeat-containing protein 5 (WDR5), RuvB-like AAA ATPase 1 (RUVBL1), RuvB-like AAA ATPase 2 (RUVBL2), transcription elongation factor SPT5 (SUPT5H), transcription elonga- tion factor SPT6 (SUPT6H), transcription elongation factor SPT4 (SUPT4H), trans- formation / transcription domain-associated protein (TRRAP) or HECT-UBA and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1). The POI may also be a protein responsible for immune system deregulation and induced autoimmunity such as Bruton tyrosine kinase (BTK), interleukin 1 receptor associated kinase 4 (IRAK4), interleukin 1 receptor associated kinase 3 (IRAK3) or mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1). The POI may also be a neurotoxic protein or protein aggregate that promote a neurodegenerative disease such as microtubule associated protein tau (MAPT), mutant huntingtin (mHtt), TAR RNA-binding protein (TARDBP), FIS RNA-binding protein (FUS), glycogen synthase kinase 3 beta (GSK3B), or synuclein alpha (SNCA). 636399-Uni Würzburg-an-1 The invention further concerns the use of the system according to the invention for checking whether a POI can act as a substrate, in particular a favorable substrate, for an enzyme selected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiq- uitinating enzyme, a kinase or a phosphatase, wherein the first recombinant pro- tein, the second recombinant protein and the ligand are brought into contact in an eukaryotic cellular environment, i. e. in eukaryotic cells, e. g. by electroporation, or an environment comprising all constituents of an eukaryotic cellular environment. An environment comprising all constituents of an eukaryotic cellular environment can be provided in the form of a lysate of eukaryotic cells. It is also possible that the first recombinant nucleic acid and the second recombinant nucleic acid are co- expressed in eukaryotic cells in cell culture followed by a treatment of the cells with the ligand. In both cases the change of cellular concentration of the POI may be monitored by measuring the amount or concentration of the reporter protein or reporter peptide in the eukaryotic cells, in the environment comprising all constitu- ents of an eukaryotic cellular environment or in medium in which the eukaryotic cells are suspended. The treatment of the cells with the ligand may occur by addi- tion of the ligand to the cells together with a substance promoting cellular uptake such as dimethyl sulfoxide (DMSO). The reporter protein or reporter peptide may be a luciferase, wherein its amount or concentration in the cells or in the medium is measured by measuring of lumines- cence after conversion of a further substrate by the luciferase. The further sub- strate may be introduced into the cells or added to the environment comprising all constituents of an eukaryotic cellular environment. It is also possible that the cells are lysed and subsequently mixed with the further substrate. The lysing of the cells and mixing with the further substrate may be performed in the medium. The invention further concerns a method for checking whether a POI can act as a substrate, in particular a favorable substrate, for an enzyme selected from an E3- ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase by use of the system according to the invention. The method may comprise the following steps: 636399-Uni Würzburg-an-1 - amplifying nucleic acid fragments coding for the enzyme, for a first binding protein of a dimerization system dimerizing the first binding protein with a second binding protein upon addition of a ligand, for the second binding protein of the dimerization system, for the reporter protein or reporter pep- tide, for the POI and optionally for the first, the second and / or the third linker by use of polymerase chain reaction (PCR), wherein the reporter protein or reporter peptide is an enzyme, in particular a luciferase, - cloning the amplified nucleic acid fragments into one or more expression vectors such that the resulting nucleic acid or nucleic acids code for the first recombinant protein and the second recombinant protein, - transformation of competent bacteria with the vector(s), selecting and ex- panding bacteria containing the vector(s) and extracting the amplified vec- tor(s), - culturing eukaryotic cells, in particular mammalian, fungal or plant cells, in cell culture, - transfection of the vector(s) into the eukaryotic cells, - expressing the first recombinant protein and the second recombinant pro- tein in the eukaryotic cells, - treating the eukaryotic cells with the ligand, optionally in combination with a cell membrane penetration promoting substance, - lysing the eukaryotic cells, adding a further substrate for the reporter pro- tein or reporter peptide and (an) additional substance(s) in case the addi- tional substance(s) is / are required for a conversion of the further substrate by the reporter protein or reporter peptide and determining whether a con- version of the further substrate changes over time, wherein a change of the conversion is an indication for the acting of the POI as a substrate for the enzyme. Treating the eukaryotic cells with the ligand may comprise electroporation. The eu- karyotic cells may be mammalian cells. The mammalian cells may be HEK-293 cells. HEK-293 cells are cells of a human embryonic kidney cell line. The first 636399-Uni Würzburg-an-1 binding protein of the dimerization system may be FKBP-rapamycin binding do- main of mTOR complex 1 (FRB) or its analog FRBT78Lor another analog of FRB, the second binding protein of the dimerization system may be 12-kDa FK506 bind- ing protein (FKBP12) or an analog thereof and the ligand may be rapamycin or an analog of rapamycin which analog is able to dimerize FRB or FRBT78Land FKBP12. Analogs of rapamycin are generally referred to as "rapalogs". The rapa- log may be AP21967 in case that the first binding protein is FRBT78L, temsirolimus, everolimus, ridaforolimus, umirolimus, or zotarolimus. Any of the before mentioned analogs of FRB and FKBP12 consists of an amino acid sequence having a se- quence identity of at least 93 % or a similarity of at least 90 % to the amino acid sequence of FRB and FKBP12, respectively, wherein the sequence similarity is calculated according to the EMBOSS needle algorithm having an open gap pen- alty of 10.0, an extended gap penalty of 0.5 and using the Blosum 62 matrix. The rapalogs (rapamycin analogues) that can be used and their IUPAC formula are as follows: 1) Temsirolimus (CCI-779): IUPAC: [(1R,2R,4S)-4-[(2R)-2- [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20- pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28- tetraen-12-yl]propyl]-2-methoxycyclohexyl] 3-hydroxy-2-(hydroxymethyl)-2- methylpropanoate 2) Everolimus (RAD001): 42-O-(2-hydroxyethyl)rapamycin IUPAC: (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3- 636399-Uni Würzburg-an-1 methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexa- methyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tet- raene-2,3,10,14,20-pentone 3) Ridaforolimus (Deforolimus, MK-8669, AP23573): IUPAC: (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-12- [(2R)-1-[(1S,3R,4R)-4-dimethylphosphoryloxy-3-methoxycyclohexyl]propan- 2-yl]-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36- dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene- 2,3,10,14,20-pentone 4) Umirolimus (Biolimus A9) IUPAC: (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-12- [(2R)-1-[(1S,3R,4R)-4-(2-ethoxyethoxy)-3-methoxycyclohexyl]propan-2-yl]- 1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-di- oxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene- 2,3,10,14,20-pentone 5) Zotarolimus (ABT-578): IUPAC: (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-19,30-dimethoxy-12-[(2R)-1-[(1S,3R,4S)-3-methoxy-4-(tetrazol-1- yl)cyclohexyl]propan-2-yl]-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4- azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20- pentone 636399-Uni Würzburg-an-1 Similarly, another rapalog, AP21967 can also be used. But it requires mutated ver- sion of FRB (FRBT78L). 6) AP21967: IUPAC: (1R,9S,12S,14S,15R,16E,18R,19S,20S,21R,23S,24E,26E,28E,30S,32S,35R)- 1,14,18,20-tetrahydroxy-12-[(2R)-1-[(1S,3R,4R)-4-hydroxy-3-methoxycyclo- hexyl]propan-2-yl]-19-methoxy-15,17,21,23,29,35-hexamethyl-30-(7-methyl- 1H-indol-3-yl)-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28- tetraene-2,3,10-trione The E3-ubiquitin ligase may be von Hibbel-Lindau E3-ubiquitin ligase (VHL), F-box protein (FBXL12) of SCF (Skp1-CUL-1-F-box protein) type E3 ubiquitin ligase complex or Cereblon E3-ubiquitin ligase (CRBN). The POI may be oncoprotein Myc, oncoprotein MycN , oncoprotein MycL, aurora kinase A (AURKA), aurora ki- nase B (AURKB), BCR-ABL fusion protein, signal transducer and activator of tran- scription 3 (STAT3), GTPase Kras (KRAS), EWS–FLI fusion protein, epidermal growth factor receptor (EGFR), Erb-b2 receptor tyrosine kinase 2 (ERBB2), ALK tyrosine kinase receptor (ALK), Wee1-like protein kinase (WEE1), catenin beta-1 (CTNNB1), Yes-associated protein 1 (YAP1), WD repeat-containing protein 5 (WDR5), RuvB-like AAA ATPase 1 (RUVBL1), RuvB-like AAA ATPase 2 (RUVBL2), transcription elongation factor SPT5 (SUPT5H), transcription elonga- tion factor SPT6 (SUPT6H), transcription elongation factor SPT4 (SUPT4H), trans- formation / transcription domain-associated protein (TRRAP), HECT-UBA and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1), Bruton tyrosine kinase (BTK), interleukin 1 receptor associated kinase 4 (IRAK4), interleukin 1 re- ceptor associated kinase 3 (IRAK3), mitogen-activated protein kinase kinase ki- nase kinase 1 (MAP4K1), microtubule associated protein tau (MAPT), mutant hun- tingtin (mHtt), TAR RNA-binding protein (TARDBP), FIS RNA-binding protein (FUS), glycogen synthase kinase 3 beta (GSK3B), or synuclein alpha (SNCA). 636399-Uni Würzburg-an-1 The reporter protein or reporter peptide may be a luciferase, in particular firefly-lu- ciferase, 19 kDa catalytic subunit of Oplophorus luciferase, in particular a lucifer- ase having sequence SEQ ID NO 1 (NanoLuc®), or a luciferase having sequence SEQ ID NO2 (HiBiT), or a fluorophore, in particular green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein(CFP), blue fluores- cent protein (BFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), far-red fluorescent protein, infra-red fluorescent protein, mNeonGFP (mNG), enhanced GFP (EGFP) or mCherry. The cell membrane penetration promoting substance may be dimethyl sulfoxide (DMSO). All features indicated in the specification are to be understood as features applica- ble to all embodiments of the invention. This means, for example, that a feature in- dicated for the system for checking whether a POI can act as a substrate for an enzyme in a eukaryotic cellular environment can also be applied to the use of the system and the method according to the invention and vice versa. The invention will be explained in more details in the following embodiments. Fig.1 shows a schematic representation of a chemically induced dimeriza- tion / chemically induced proximity of two different proteins or do- mains. Fig.2 shows the schematic representation of the rapamycin induced prox- imity between E3-ubiquitin ligase and a POI as a target. Fig.3 shows a vector map of a construct for transient expression of VHL- FRB. Fig.4 shows a vector map of a construct for transient expression of CRBN- FRB. 636399-Uni Würzburg-an-1 Fig.5 shows a vector map of a construct for transient expression of FRB. Fig.6 shows a vector map of a construct for transient expression of WDR5- NanoLuc®-FKBP12. Fig.7 shows an immunoblot of HEK293 cells transiently transfected with SFFV promoter containing WDR5-FKBP12-luciferase and VHL-FRB or FRB constructs in the ratio of 1:1 or 1:10 (FKBP12: FRB) which cells were treated with 10 nM rapamycin in a solution or the solution without rapamycin for 6 hours, wherein vinculin is used as a control for checking whether each lane of the gel has been uniformly loaded. Fig.8 shows WDR5 levels / concentrations based on luciferase measure- ment of HEK293 cells transiently expressing WDR5-FKBP12- luciferase and VHL-FRB, CRBN-FRB or FRB at various time points after rapamycin treatment. Fig.9 shows an immunoblot of HEK-293 cells transiently expressing WDR5-FKBP12-luciferase and VHL-FRB or CRBN-FRB or FRB treated with rapamycin in a solution or the solution without rapamycin for 6 hours, wherein vinculin is used as a control for checking whether each lane of the gel has been uniformly loaded. Fig.10 shows WDR5 concentrations / levels based on luciferase measure- ment of HEK-293 cells transiently expressing WDR5-FKBP12- luciferase, wherein all lysine residues in the FKBP12 and in the lucif- erase have been replaced by arginine, and VHL-FRB or CRBN-FRB or FRB at various time points after rapamycin treatment. Fig.11 shows an immunoblot of HEK-293 cells transiently expressing WDR5-FKBP12-luciferase, wherein all lysine residues in FKBP12 636399-Uni Würzburg-an-1 and in the luciferase have been replaced by arginine residues, and VHL-FRB, CRBN-FRB or FRB treated with rapamycin in a solution or the solution without rapamycin for 6 hours, wherein vinculin is used as a control for the uniformly loading of each lane of the gel. Fig.12 shows an immunoblot of HEK-293 cells transiently expressing AURKA-luciferase-FKBP12 and VHL-FRB or CRBN-FRB or FRB, treated with vehicle or rapamycin for 6 hours. Vinculin is used as load- ing control. Fig.13 shows AURKA concentrations / levels based on luciferase measure- ment of HEK-293 cells transiently expressing AURKA-luciferase- FKBP12 and VHL-FRB or CRBN-FRB or FRB at various timepoints after rapamycin treatment. Fig.14 shows an immunoblot of HEK-293 cells transiently expressing WDR5- luciferase-FKBP12 (K-less FKBP12 and luciferase) and FBXL12-FRB or FRB, treated with vehicle or rapamycin for 8 hours. Vinculin is used as loading control. Fig.15 shows WDR5 concentrations / levels based on luciferase measurement of HEK-293 cells transiently expressing WDR5-luciferase-FKBP12 (K- less FKBP12 and luciferase) and FBXL12-FRB or FRB after 8 hours of rapamycin treatment. Fig.16 shows WDR5 concentrations / levels based on luciferase measurement of HEK-293 cells transiently expressing WDR5-luciferase-FKBP12 and VHL-FRB or FBXL12-FRB or FRB at various timepoints after ra- pamycin treatment. Fig.17 shows WDR5 concentrations / levels based on kinetic luciferase meas- urement of HEK-293 cells transiently expressing WDR5-luciferase- FKBP12 (K-less FKBP12 and luciferase) and FBXL12-FRB or FRB, measured every15-30 minutes for 19 hours after rapamycin treatment. 636399-Uni Würzburg-an-1 Fig.18 shows WDR5 concentrations / levels based on luciferase measurement of HEK-293 cells transiently expressing WDR5-luciferase-FKBP12 or luciferase-WDR5-FKBP12 and FBXL12-FRB or FRB after 9 hours of rapamycin treatment. Fig.19 shows immunoblots of HEK-293 cells transiently expressing lucifer- ase-WDR5-FKBP12 with indicated linker length (between WDR5 and FKBP12) and FBXL12-FRB or FRB, treated with vehicle or rapamycin for 6 hours. Vinculin is used as loading control. Fig.20 shows WDR5 concentrations / levels based on luciferase measurement of HEK-293 cells transiently expressing luciferase-WDR5-FKBP12 with indicated linker length (between WDR5 and FKBP12) and FBXL12-FRB or FRB after 8 hours of rapamycin treatment. Fig.21 shows an estimated time line for the performance of the method ac- cording to the invention. Embodiments: In the embodiments and in the figures "E3 ligase" and "E3-ligase" always means "E3-ubiquitin ligase" and "target" means the POI. 1. Exemplary experimental steps for CRISPR knock-in: 1) Design of knock-in tag and generation of the homology directed repair (HDR) template plasmid. Some exemplary designs of HDR templates are as follows: a) N-terminal target tagging with FKBP12 and NanoLuc®: LHA – Start codon – Selection Marker – P2A – FKBP12 – linker – NanoLuc® – linker – RHA, 636399-Uni Würzburg-an-1 wherein LHA is a left homology arm which consists of the genomic sequence before the start codon of the target protein, RHA is a right homology arm which consists of the genomic sequence after the start codon of the target protein, and P2A is a 2A self-cleaving peptide. b) C-terminal target tagging with FKBP12 and NanoLuc®: LHA – linker – NanoLuc® – linker – FKBP12 – P2A – Selection Marker – Stop codon – RHA, wherein LHA is a left homology arm which consists of the genomic sequence before the stop codon of the target protein, RHA is a right homology arm which consists of the genomic sequence after the stop codon of the target protein, and P2A is 2A self-cleaving peptide. In the above example for FKBP12 knock-in, the position of NanoLuc® and FKBP12 can also be interchanged. c) N-terminal E3-ligase tagging with FRB: LHA – Start codon – Selection Marker – P2A – FRB – linker – RHA, wherein LHA is a left homology arm which consists of the genomic sequence before the start codon of the E3-ligase, RHA is a right ho- mology arm which consists of genomic sequence after the start codon of the E3-ligase, and P2A is 2A self-cleaving peptide. d) C-terminal E3-ligase tagging with FRB: LHA – linker – FRB – P2A – Selection Marker – Stop codon – RHA, 636399-Uni Würzburg-an-1 wherein LHA is a left homology arm which consists of the genomic sequence before the stop codon of the E3-ligase, RHA is a right ho- mology arm which consists of genomic sequence after the stop codon of the E3-ligase, and P2A is 2A self-cleaving peptide. 2) Design of the sgRNA and sgRNA cloning in any Cas9-sgRNA vector, such as PX458 sold by the company Addgene as Plasmid #48138 sgRNA should ideally be designed for the specific terminus namely around the start codon for N-terminal tagging or around the stop codon for C-terminal tag- ging. The sgRNA sequence must be followed by the PAM site i.e., NGG for sense strand or CCN for the antisense strand. 3) Transfect the cells with both sgRNA and HDR template plasmids. 4) Post 72 hours of transfection, select the cells using a selection marker introduced into the genomic locus using HDR template. 5) Pick the single colony of the selected cells either by serial dilution, FACS sorting or cloning ring. Expand the colony of singe cell. 6) Identify the positive clones with homozygous knock-in using genotypic PCR or western blot (WB). For genotypic PCR, extract the genomic DNA from the clones and perform PCR using screening primers that bind outside the homology arms. Confirm the correct integration by Sanger sequencing of the PCR product span- ning the genomic integration site. For WB, probe the whole membrane with the respective antibodies for target protein or E3-ligase. Successful knock-in should result in increase of the size of PCR product or molecular weight of protein as compared to the PCR product or protein molecular weight from the naive cells. 636399-Uni Würzburg-an-1 2. Generation of VHL-FRB, CRBN-FRB, FRB and WDR5-NanoLuc®-FKBP12 constructs i. Primer pairs were designed with appropriate restriction sites, linkers, start and stop codon. The primers were ordered from Merck KGaA, Deutschland, Sigma-Aldrich to amplify VHL, CRBN, FRB, WDR5, NanoLuc® luciferase and FKBP12 segments of the constructs. ii. PCR amplification of different segments were carried out with PhusionTMpol- ymerase using following templates: a. VHL: complementary DNA (cDNA) b. CRBN: plasmid from addgene c. FRB: complementary DNA (cDNA) d. WDR5: plasmid from Promega e. NanoLuc®: custom double-stranded DNA fragments (gBlock, ordered from IDT integrated DNA technologies) f. FKBP12: plasmid from addgene iii. Agarose gel electrophoresis of the PCR products were carried out to check the size of the products. iv. The PCR products with expected band size were gel purified. For this the PCR fragment was cut-out from the agarose gel and extracted using the GeneJET Gel Extraction Kit from the company Thermo Fisher Scientific ac- cording to the manufacturer’s instructions. v. Any eukaryotic expression vector can be used for the preparation of the FKBP12 and FRB constructs. In this example, FRB constructs were cloned into pRRLSin.cPPT.WPRE vector with SFFV (Spleen Focus Forming Virus) promoter and Hygromycin as resistance marker. Likewise, FKBP12 construct was cloned into pRRLSin.cPPT.WPRE vector with SFFV promoter and Puro- mycin as resistance marker. vi. The PCR amplified fragments and vector backbone (pRRLSin.cPPT:WPRE) were digested with appropriate restriction enzymes. vii. The digested PCR fragments and vector backbone were gel purified. 636399-Uni Würzburg-an-1 viii. The concentration of the digested products was measured using Multiscan Ascent Spectrofluorometer NanoDrop 1000 from Thermo Fisher Scientific. ix. The digested PCR fragments (inserts) and vector backbone were ligated in a molar ratio of 3:1 (insert : vector backbone). x. The ligated constructs were transformed into chemically competent E. coli XL1 blue and bacteria were streaked onto LB-agar plates with ampicillin (pRRL vector consists of ampicillin resistance). xi. Single colonies of transformed bacteria were propagated in LB medium with ampicillin. Plasmid was isolated and purified from the bacterial culture using PureLink HiPure Plasmid Maxiprep Kit according to the manufacturer’s pro- tocol. xii. The sequence of the plasmid was confirmed by Sanger sequencing. xiii. The sequence of the fragments of different constructs are as follows: a. VHL-FRB VHL-linker-FRB: SEQ ID NO 9 ATGCCCCGGAGGGCGGAGAACTGGGACGAGGCCGAGGTAGGC GCGGAGGAGGCAGGCGTCGAAGAGTACGGCCCTGAAGAAGAC GGCGGGGAGGAGTCGGGCGCCGAGGAGTCCGGCCCGGAAGA GTCCGGCCCGGAGGAACTGGGCGCCGAGGAGGAGATGGAGG CCGGGCGGCCGCGGCCCGTGCTGCGCTCGGTGAACTCGCGC GAGCCCTCCCAGGTCATCTTCTGCAATCGCAGTCCGCGCGTCG TGCTGCCCGTATGGCTCAACTTCGACGGCGAGCCGCAGCCCTA CCCAACGCTGCCGCCTGGCACGGGCCGCCGCATCCACAGCTA CCGAGGTCACCTTTGGCTCTTCAGAGATGCAGGGACACACGAT GGGCTTCTGGTTAACCAAACTGAATTATTTGTGCCATCTCTCAAT GTTGACGGACAGCCTATTTTTGCCAATATCACACTGCCAGTGTA TACTCTGAAAGAGCGATGCCTCCAGGTTGTCCGGAGCCTAGTC AAGCCTGAGAATTACAGGAGACTGGACATCGTCAGGTCGCTCT ACGAAGATCTGGAAGACCACCCAAATGTGCAGAAAGACCTGGA GCGGCTGACACAGGAGCGCATTGCACATCAACGGATGGGAGAT GAATTCGGGAGCTCCGGTGGGAGCTCCGGTATCCTCTGGCATG 636399-Uni Würzburg-an-1 AGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTT GGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCT TGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAAC ATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAG AGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCT CACCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCT CAAAGTGA b. CRBN-FRB CRBN-linker-FRB: SEQ ID NO 10 ATGGCCGGCGAAGGAGATCAGCAGGACGCTGCGCACAACATG GGCAACCACCTGCCGCTCCTGCCTGCAGAGAGTGAGGAAGAA GATGAAATGGAAGTTGAAGACCAGGATAGTAAAGAAGCCAAAAA ACCAAACATCATAAATTTTGACACCAGTCTGCCGACATCACATA CATACCTAGGTGCTGATATGGAAGAATTTCATGGCAGGACTTTG CACGATGACGACAGCTGTCAGGTGATTCCAGTTCTTCCACAAGT GATGATGATCCTGATTCCCGGACAGACATTACCTCTTCAGCTTT TTCACCCTCAAGAAGTCAGTATGGTGCGGAATTTAATTCAGAAA GATAGAACCTTTGCTGTTCTTGCATACAGCAATGTACAGGAAAG GGAAGCACAGTTTGGAACAACAGCAGAGATATATGCCTATCGA GAAGAACAGGATTTTGGAATTGAGATAGTGAAAGTGAAAGCAAT TGGAAGACAAAGGTTCAAAGTCCTTGAGCTAAGAACACAGTCAG ATGGAATCCAGCAAGCTAAAGTGCAAATTCTTCCCGAATGTGTG TTGCCTTCAACCATGTCTGCAGTTCAATTAGAATCCCTCAATAAG TGCCAGATATTTCCTTCAAAACCTGTCTCAAGAGAAGACCAATG TTCATATAAATGGTGGCAGAAATACCAGAAGAGAAAGTTTCATT GTGCAAATCTAACTTCATGGCCTCGCTGGCTGTATTCCTTATAT GATGCTGAGACCTTAATGGACAGAATCAAGAAACAGCTACGTGA ATGGGATGAAAATCTAAAAGATGATTCTCTTCCTTCAAATCCAAT AGATTTTTCTTACAGAGTAGCTGCTTGTCTTCCTATTGATGATGT ATTGAGAATTCAGCTCCTTAAAATTGGCAGTGCTATCCAGCGAC 636399-Uni Würzburg-an-1 TTCGCTGTGAATTAGACATTATGAATAAATGTACTTCCCTTTGCT GTAAACAATGTCAAGAAACAGAAATAACAACCAAAAATGAAATAT TCAGTTTATCCTTATGTGGGCCGATGGCAGCTTATGTGAATCCT CATGGATATGTGCATGAGACACTTACTGTGTATAAGGCTTGCAA CTTGAATCTGATAGGCCGGCCTTCTACAGAACACAGCTGGTTTC CTGGGTATGCCTGGACTGTTGCCCAGTGTAAGATCTGTGCAAG CCATATTGGATGGAAGTTTACGGCCACCAAAAAAGACATGTCAC CTCAAAAATTTTGGGGCTTAACGCGATCTGCTCTGTTGCCCACG ATCCCAGACACTGAAGATGAAATAAGTCCAGACAAAGTAATACT TTGCTTGACGCGTGGGAGCTCCGGTGGGAGCTCCGGTATCCTC TGGCATGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTT TGTACTTTGGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCT GGAGCCCTTGCATGCTATGATGGAACGGGGCCCCCAGACTCTG AAGGAAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGA GGCCCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTC c. FRB: SEQ ID NO 11 ATGATCCTCTGGCATGAGATGTGGCATGAAGGCCTGGAAGAGG CATCTCGTTTGTACTTTGGGGAAAGGAACGTGAAAGGCATGTTT GAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCCCC CAGACTCTGAAGGAAACATCCTTTAATCAGGCCTATGGTCGAGA TTTAATGGAGGCCCAAGAGTGGTGCAGGAAGTACATGAAATCA GGGAATGTCAAGGACCTCACCCAAGCCTGGGACCTCTATTATC ATGTGTTCCGACGAATCTCAAAGTGA d. WDR5-NanoLuc®-FKBP12 WDR5-linker- linker-FKBP12: SEQ ID NO 12 636399-Uni Würzburg-an-1 ATGGCGACGGAGGAGAAGAAGCCCGAGACCGAGGCCGCCAGA GCACAGCCAACCCCTTCGTCATCCGCCACTCAGAGCAAGCCTA CACCTGTGAAGCCAAACTATGCTCTAAAGTTCACCCTTGCTGGC CACACCAAAGCAGTGTCCTCCGTGAAATTCAGCCCGAATGGAG AGTGGCTGGCAAGTTCATCTGCTGATAAACTTATTAAAATTTGG GGCGCGTATGATGGGAAATTTGAGAAAACCATATCTGGTCACAA GCTGGGAATATCCGATGTAGCCTGGTCGTCAGATTCTAACCTTC TTGTTTCTGCCTCAGATGACAAAACCTTGAAGATATGGGACGTG AGCTCGGGCAAGTGTCTGAAAACCCTGAAGGGACACAGTAATT ATGTCTTTTGCTGCAACTTCAATCCCCAGTCCAACCTTATTGTCT CAGGATCCTTTGACGAAAGCGTGAGGATATGGGATGTGAAAAC AGGGAAGTGCCTCAAGACTTTGCCAGCTCACTCGGATCCAGTC TCGGCCGTTCATTTTAATCGTGATGGATCCTTGATAGTTTCAAGT AGCTATGATGGTCTCTGTCGCATCTGGGACACCGCCTCAGGCC AGTGCCTGAAGACGCTCATCGATGACGACAACCCCCCCGTGTC TTTTGTGAAGTTCTCCCCGAACGGCAAATACATCCTGGCCGCCA CGCTGGACAACACTCTGAAGCTCTGGGACTACAGCAAGGGGAA GTGCCTGAAGACGTACACTGGCCACAAGAATGAGAAATACTGC ATATTTGCCAATTTCTCTGTTACTGGTGGGAAGTGGATTGTGTCT GGCTCAGAGGATAACCTTGTTTACATCTGGAACCTTCAGACGAA AGAGATTGTACAGAAACTACAAGGCCACACAGATGTCGTGATCT CAACAGCTTGTCACCCAACAGAAAACATCATCGCCTCTGCTGCG CTAGAAAATGACAAAACAATTAAACTGTGGAAGAGTGACTGCGA ATTCGGGAGCTCCGGTGGGAGCTCCGGTGTCTTCACACTCGAA GATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGG ACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAAT CTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCG GTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTAT GAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTT TTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATC CTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACA TGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTT 636399-Uni Würzburg-an-1 CGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGC GCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTA CACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGG GACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGT GATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGG TCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTG CCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGT xiv. Vector maps for the constructs are given in Figs.3 to 6. In the vector map given in Fig.6 NanoLuc® is given as "Luc". 3. Transfection and rapamycin treatment i. After the generation of vectors, HEK293 cells were cultured appropriately prior to assay. ii. Medium was removed from cell plates by aspiration and cells were tryp- sinized to dissociate from the cell culture dish bottom. iii. Trypsin was neutralized using cell culture medium, cells were counted and seeded in the density of 4 million cells into 10 cm dishes in cell culture me- dium. iv. The cells were allowed to attach and recover for at least 6 hours at 37 °C, 5% CO2. v. The SFFV promoter containing constructs of FKBP12, and FRB were trans- fected in the weight : weight ratios of 1:1, 1:10 and 1:100(FKBP12 : FRB). For each of these transfection ratios the following combinations of plasmid constructs were added in separate tubes to 700 µl of Opti-MEM each: 636399-Uni Würzburg-an-1 a. VHL-FRB (8 µg) and WDR5-NanoLuc®-FKBP12 (0.8 µg) b. CRBN-FRB (8 µg) and WDR5-NanoLuc®-FKBP12 (0.8 µg) c. FRB (8 µg) and WDR5-NanoLuc®-FKBP12 (0.8 µg) vi. 30 µl of polyethylenimine (PEI) was added to 700 µl Opti-MEM (per sample), mixed well (vortexed), centrifuged, and incubated at RT for 5 minutes. vii. The plasmid mixture was added to the PEI mixture, mixed by pipetting up and down and left at RT for 20 min. viii. The transfection mixture was added to 10 cm plates with attached cells. ix. The proteins were expressed for at least 18 hours. x. After 18-24 hours, the cells were resuspended in 12 ml of media after tryp- sinization. xi. 40 µl of DMEM was added in all the wells of black 96 well plate and 30 µl of cell suspension per well were seeded into 3x5x2 wells of 96-well plate per transfection condition (for luciferase measurement). The total volume of cell suspension in 96 well plate is 70 µl now. For 96-well plate, 3 wells will be DMSO control, and 3 wells will be treated with 10 nM Rapamycin for 5 differ- ent time points (1h, 2h, 4h, 6h and 8h). Similarly, 2 x 1ml of cell suspension were seeded into 6-well plates (contain- ing 1 ml media in each well) per transfection condition (for western blot, WB). The total volume of cell suspension in 6-well plate is 2 ml now. For 6-well plate only 6h timepoint was used for treatment. xii. Plates were shaken back and forth to get even seeding. The plates were left in incubator so that the cells attach overnight. xiii. Next day, the cells were treated at different time points and harvested to- gether. xiv. For treatment, 10 nM final concentration of Rapamycin or DMSO was used. a. Rapamycin was dissolved in DMSO at a concentration of 10 mM as stock solution. The 10 mM Rapamycin was diluted in DMSO to reach concentration of 10 µM. b. For 96 well plate, 2.4 µl DMSO or 10 µM Rapamycin was added in 700 µl warm culture media.30 µl of the resulting solution was added to the 636399-Uni Würzburg-an-1 cells in 70 µl media to get the final concentration of 10 nM Rapamycin. The Rapamycin and DMSO solution in media were prepared just be- fore their addition. c. For 6-well plate, 2 µl of DMSO or 2 µl 10 µM Rapamycin was added to the cells in 2 ml media to get the final concentration of 10 nM Ra- pamycin. xv. For luciferase assay, NanoGlo® Luciferase assay reagent with the substrate (Promega) was added 1:1 with the cells with media, mixed properly, incu- bated for 5-10 minutes at RT with shaking and luminescence was measured (according to manufacturer’s protocol). xvi. For WB, cells were washed 2 x with ice-cold PBS and harvested in RIPA buffer. BCA assay was performed to determine the protein concentration and the samples were prepared in Laemmli buffer. Equal protein amount per sam- ples were run in PAGE, transferred into PVDF membrane, blocked with 5% milk, and finally probed against WDR5, VHL, CRBN and Vinculin antibodies. The WB results for WDR5, VHL and Vinculin and transfection ratios of 1:1 and 1:10 (FKBP12 : FRB) are shown in Fig.7. It has been found that the E3- ligase (FRB) should be in excess to the target protein (FKBP12) counterpart. With the transfection ratio of 1:1 no degradation of WDR5 fusion protein in the presence of Rapamycin was observed in the WB whereas with ratio 1:10 (FKBP12 : FRB) degradation was observed. A similar degradation of WDR5 fusion protein was also observed with a transfection ratio of 1:100 (FKBP12 : FRB). It is assumed that little degradation of WDR5 fusion protein even occurs with the transfection ratio of 1:1 but cannot be observed in WB. Results of the luciferase assay obtained with a transfection ratio 1:10 (FKBP12 : FRB) are shown in Fig.8. WB results obtained with a transfection ratio of 1:10 (FKBP12 : FRB) are shown in Fig.9. The results show the effec- tive degradation of WDR5 by VHL but not by CRBN. This is in line with pre- vious findings obtained with PROTACs indicating that WDR5 can act as a substrate for VHL but not for CRBN. 636399-Uni Würzburg-an-1 4. Modified system for excluding ubiquitination of the tag Both FKBP12 and NanoLuc® amino acid sequences comprise 7 and 8 lysine resi- dues (K, shown in bold in the sequences below), respectively. Thus, the above as- say could not distinguish between ubiquitination of the target protein and of FKBP12 and / or NanoLuc®. For restricting the potential ubiquitination to the target protein the above assay has been modified by replacing the lysine residues by arginine resi- dues. The canonical sequence of the FKBP12 and NanoLuc® are as follows: a. FKBP12 (107 amino acids): SEQ ID NO 13 GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHP GIIPPHATLVFDVELLKLE b. NanoLuc® (170 amino acids): SEQ ID NO 1 VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVIL HYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIID ERLINPDGSLLFRVTINGVTGWRLCERILA Careful analysis of the publicly available crystal structure of these proteins showed that all the lysine residues are present on the surface of these proteins. Such lysine residues may be potential ubiquitination sites for the E3-ligase in the assay. Such ubiquitination and eventual degradation of the fusion protein might give false posi- tive result in the assay. Thus, to prevent such false positive results and to improve the assay further, a lysine less (K-less) version of FKBP12 and NanoLuc® construct was generated. For this 636399-Uni Würzburg-an-1 all the lysine residues from both protein tags, FKBP12 and NanoLuc® were replaced with arginine. Such a K-less construct reliably prevents degradation of target protein only due to ubiquitination of FKBP12 and NanoLuc® sequences in the second re- combinant protein. Nucleic acids coding for the K-less versions of FKBP12 and NanoLuc® were ordered as custom double-stranded DNA fragments (gBlock) from the company IDT integrated DNA technologies. The amino acid sequences of the K-less versions of FKBP12 and NanoLuc®, in which replaced arginine residues are shown as R in bold, are as follows: a. FKBP12 (107 amino acids): SEQ ID NO 14 GVQVETISPGDGRTFPRRGQTCVVHYTGMLEDGRRFDSSRDRNR PFRFMLGRQEVIRGWEEGVAQMSVGQRARLTISPDYAYGATGHP GIIPPHATLVFDVELLRLE b. NanoLuc (170 amino acids): SEQ ID NO 15 VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLRIDIHVIIPYEGLSGDQMGQIERIFRVVYPVDDHHFRVIL HYGTLVIDGVTPNMIDYFGRPYEGIAVFDGRRITVTGTLWNGNRIID ERLINPDGSLLFRVTINGVTGWRLCERILA Then the WDR5-NanoLuc-FKBP12 (K-less) construct was generated (similar to the wild-type with the linker in between the proteins), and the assay was repeated as for WDR5-NanoLuc-FKBP12 wild-type with VHL-FRB, CRBN-FRB and FRB. The results from the Nanoluciferase assay and the WB are shown in Fig.10 and Fig.11. Fig.10 shows WDR5 levels based on luciferase measurement of HEK293 cells tran- siently expressing WDR5-FKBP12-luciferase (K-less FKBP12 and luciferase) and VHL-FRB or CRBN-FRB or FRB at various timepoints after rapamycin treatment. Fig.11 shows an immunoblot of HEK293 cells transiently expressing WDR5- FKBP12-luciferase (K-less FKBP12 and luciferase) and VHL-FRB or CRBN-FRB 636399-Uni Würzburg-an-1 or FRB, treated with vehicle or rapamycin for 6 hours. Vinculin is used as loading control. The results show the effective degradation of WDR5 by VHL but not by CRBN. The assay confirms that it was the compatibility of WDR5 with VHL that led to ubiquiti- nation of WDR5 and finally proteasomal degradation of the WDR5-NanoLuc- FKBP12 fusion protein in the first assay and not compatibility of FKBP12 and / or NanoLuc® with VHL. Once a library of E3-ubiquitin ligases with tags is cloned, a tentative timeline for the assay with any target protein is given in Fig.21. 5. System with AURKA as POI CRBN-based PROTACs have been shown to efficiently degrade mitotic serine / thre- onine kinase, Aurora-A (AURKA). An AURKA-NanoLuc-FKBP12 construct was made similar to that of WDR5-NanoLuc-FKBP12. The amino acid sequence of human Aurora-A (SEQ ID NO 24) is as follows: MDRSKENCISGPVKATAPVGGPKRVLVTQQFPCQNPLPVNSGQAQRVLCPSNS SQRVPLQAQKLVSSHKPVQNQKQKQLQATSVPHPVSRPLNNTQKSKQPLPSAP ENNPEEELASKQKNEESKKRQWALEDFEIGRPLGKGKFGNVYLAREKQSKFILAL KVLFKAQLEKAGVEHQLRREVEIQSHLRHPNILRLYGYFHDATRVYLILEYAPLGT VYRELQKLSKFDEQRTATYITELANALSYCHSKRVIHRDIKPENLLLGSAGELKIAD FGWSVHAPSSRRTTLCGTLDYLPPEMIEGRMHDEKVDLWSLGVLCYEFLVGKPP FEANTYQETYKRISRVEFTFPDFVTEGARDLISRLLKHNPSQRPMLREVLEHPWIT ANSSKPSNCQNKESASKQS The nucleotide sequence of the AURKA-NanoLuc-FKBP12-fragment of AURKA- NanoLuc-FKBP12 construct (SEQ ID NO 25) is as follows: 636399-Uni Würzburg-an-1 AURKA-NanoLuc-FKBP12: AURKA-linker-NanoLuc- ATGGACCGATCTAAAGAAAACTGCATTTCAGGACCTGTTAAGGCTACAGCTCC AGTTGGAGGTCCAAAACGTGTTCTCGTGACTCAGCAATTTCCTTGTCAGAATC CATTACCTGTAAATAGTGGCCAGGCTCAGCGGGTCTTGTGTCCTTCAAATTCTT CCCAGCGCGTTCCTTTGCAAGCACAAAAGCTTGTCTCCAGTCACAAGCCGGT TCAGAATCAGAAGCAGAAGCAATTGCAGGCAACCAGTGTACCTCATCCTGTCT CCAGGCCACTGAATAACACCCAAAAGAGCAAGCAGCCCCTGCCATCGGCACC TGAAAATAATCCTGAGGAGGAACTGGCATCAAAACAGAAAAATGAAGAATCAA AAAAGAGGCAGTGGGCTTTGGAAGACTTTGAAATTGGTCGCCCTCTGGGTAA AGGAAAGTTTGGTAATGTTTATTTGGCAAGAGAAAAGCAAAGCAAGTTTATTCT GGCTCTTAAAGTGTTATTTAAAGCTCAGCTGGAGAAAGCCGGAGTGGAGCATC AGCTCAGAAGAGAAGTAGAAATACAGTCCCACCTTCGGCATCCTAATATTCTTA GACTGTATGGTTATTTCCATGATGCTACCAGAGTCTACCTAATTCTGGAATATGC ACCACTTGGAACAGTTTATAGAGAACTTCAGAAACTTTCAAAGTTTGATGAGCA GAGAACTGCTACTTATATAACAGAATTGGCAAATGCCCTGTCTTACTGTCATTC GAAGAGAGTTATTCATAGAGACATTAAGCCAGAGAACTTACTTCTTGGATCAGC TGGAGAGCTTAAAATTGCAGATTTTGGGTGGTCAGTACATGCTCCATCTTCCA GGAGGACCACTCTCTGTGGCACCCTGGACTACCTGCCCCCTGAAATGATTGA AGGTCGGATGCATGATGAGAAGGTGGATCTCTGGAGCCTTGGAGTTCTTTGC TATGAATTTTTAGTTGGGAAGCCTCCTTTTGAGGCAAACACATACCAAGAGACC TACAAAAGAATATCACGGGTTGAATTCACATTCCCTGACTTTGTAACAGAGGGA GCCAGGGACCTCATTTCAAGACTGTTGAAGCATAATCCCAGCCAGAGGCCAAT GCTCAGAGAAGTACTTGAACACCCCTGGATCACAGCAAATTCATCAAAACCAT CAAATTGCCAAAACAAAGAATCAGCTAGCAAACAGTCTCTCGAGGGGAGCTC CGGTGGGAGCTCCGGTGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCG ACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTC CAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCC TGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAA GGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGT ACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTA 636399-Uni Würzburg-an-1 ATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAG GCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAA CGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTG ACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGC GTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCG GGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGA GGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTG ACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCA CCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATGA An assay was performed with VHL-FRB, CRBN-FRB, and FRB. In contrast to WDR5, both E3-ligases CRBN and VHL led to the productive degradation of Aurora- A. The results from immunoblot and nanoluciferase assay are shown in Fig.12 and Fig.13. 6. System with FBXL12 as E3 ubiquitin ligase FBXL12 is currently not used for PROTACs. An FBXL12-FRB construct was gener- ated similar to the VHL-FRB and CRBN-FRB with the linker between E3-ligase and FRB. The amino acid sequence of human FBXL12 (SEQ ID NO 26) is as follows: MATLVELPDSVLLEIFSYLPVRDRIRISRVCHRWKRLVDDRWLWRHVDLTLYTMRP KVMWHLLRRYMASRLHSLRMGGYLFSGSQAPQLSPALLRALGQKCPNLKRLCL HVADLSMVPITSLPSTLRTLELHSCEISMAWLHKQQDPTVLPLLECIVLDRVPAFR DEHLQGLTRFRALRSLVLGGTYRVTETGLDAGLQELSYLQRLEVLGCTLSADSTL LAISRHLRDVRKIRLTVRGLSAPGLAVLEGMPALESLCLQGPLVTPEMPSPTEILSS CLTMPKLRVLELQGLGWEGQEAEKILCKGLPHCMVIVRACPKESMDWW 636399-Uni Würzburg-an-1 The nucleotide sequence of the FBXL12-FRB-fragment of FBXL12-FRB (SEQ ID NO 27) construct is as follows: FBXL12-FRB FBXL12-linker-FRB ATGGCGACTTTGGTCGAACTGCCGGACTCGGTCCTGCTCGAGATCTTCTCTTA CCTCCCGGTACGGGACCGGATCCGCATCTCCAGGGTCTGTCACCGCTGGAA GAGGCTGGTGGACGACCGGTGGCTGTGGCGACATGTCGACCTGACGCTCTA CACGATGCGACCTAAAGTCATGTGGCACCTCCTTCGAAGGTACATGGCATCCC GGCTCCATTCCCTGCGGATGGGTGGCTACCTGTTCTCTGGCTCCCAGGCCCC CCAGTTGTCCCCTGCTCTGTTGAGAGCCCTGGGCCAGAAGTGCCCCAACCT GAAGCGCCTCTGCCTGCACGTGGCCGACCTGAGCATGGTGCCCATCACCAG CCTGCCCAGCACCTTGAGGACCCTGGAGCTGCACAGCTGCGAGATCTCCAT GGCCTGGCTCCACAAGCAGCAGGACCCCACCGTGCTGCCCCTGCTTGAATG CATCGTGCTGGACCGCGTCCCCGCCTTCCGTGACGAGCACCTGCAGGGCCT GACGCGCTTCCGGGCCTTGCGCTCGCTGGTGCTGGGTGGTACCTACCGTGT GACCGAGACAGGGCTGGATGCTGGCCTGCAGGAGCTCAGCTATCTGCAGAG GCTTGAGGTGCTGGGCTGCACCCTGTCTGCCGACAGCACCCTGCTGGCCAT CAGCCGCCACCTCCGAGATGTGCGCAAGATCCGGCTGACCGTGAGGGGCCT CTCTGCCCCTGGCCTGGCTGTGCTGGAGGGAATGCCGGCCCTGGAGAGTCT GTGCCTGCAGGGTCCCCTCGTCACCCCAGAAATGCCCTCCCCCACTGAAATC CTCTCCTCCTGCCTCACTATGCCCAAGCTCAGAGTCCTTGAGCTGCAGGGGC TGGGGTGGGAGGGTCAGGAGGCGGAGAAGATCCTGTGTAAGGGGCTGCCC CACTGTATGGTCATCGTCAGGGCTTGCCCCAAAGAGTCTATGGACTGGTGGAT GACGCGTGGGAGCTCCGGTGGGAGCTCCGGTATCCTCTGGCATGAGATGTG GCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAACGTG AAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCC CCCAGACTCTGAAGGAAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATG GAGGCCCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACC TCACCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCTCAAAGTGA 636399-Uni Würzburg-an-1 An assay was performed for WDR5-NanoLuc-FKBP12 (K-less FKBP12 and lucifer- ase) with FBXL12-FRB and FRB. The results from immunoblot and luciferase assay are shown in Fig.14 and Fig.15. The assays showed robust degradation of WDR5 fusion protein. Furthermore, a direct comparison of WDR5 degradation via VHL and FBXL12 was performed with a time course experiment. The result as shown in Fig. 16 displayed a superior activity of FBXL12. 7. Kinetic measurement In the above assays POI degradation was demonstrated by immunoblotting or by luciferase activity measurement after cell lysis. Since it is not only relevant how com- plete target degradation is, but also how fast, a workflow for kinetic analyses was established. For this purpose, the luciferase substrate used for lytic measurement was replaced with endurazine as a substrate which is continuously taken up and activated by the cells. The assay was then performed with WDR5-NanoLuc-FKBP12 (K-less) and FBXL12-FRB or FRB and luciferase activity was measured every 15 minutes for 6 hours followed by every 30 minutes in the presence or absence of rapamycin. Rapamycin-mediated dimerization with FBXL12 induced a robust and durable degradation of WDR5, reaching a maximum after 5 hours as shown in Fig. 17. 8. Effect of linker length on target protein degradation For investigating the effect of steric arrangement between the target protein, WDR5 and the FKBP12 tag, the length of the linker between WDR5 and FKBP12 was var- ied in NanoLuc-WDR5-FKBP12 constructs. In constructs described above the linker between components of the POI fusion protein, i. e. between POI and NanoLuc and between NanoLuc and FKBP12, was 2xGSSG. The nucleotide sequences of the NanoLuc-WDR5-FKBP12-fragments of NanoLuc- WDR5-FKBP12 constructs with various linker length between WDR5 and FKBP12 are as follows: 636399-Uni Würzburg-an-1 a. 0xGSSG; NanoLuc-WDR5-FKBP12 NanoLuc-linker- FKBP12 (SEQ ID NO 28) ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCT ACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAA TCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCG ACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTAC GCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTC GACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTAT CGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATC AACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGGAGCTCC GGTGGGAGCTCCGGTGAATTCGCGACGGAGGAGAAGAAGCCCGAGACCGA GGCCGCCAGAGCACAGCCAACCCCTTCGTCATCCGCCACTCAGAGCAAGCC TACACCTGTGAAGCCAAACTATGCTCTAAAGTTCACCCTTGCTGGCCACACCA AAGCAGTGTCCTCCGTGAAATTCAGCCCGAATGGAGAGTGGCTGGCAAGTTC ATCTGCTGATAAACTTATTAAAATTTGGGGCGCGTATGATGGGAAATTTGAGAA AACCATATCTGGTCACAAGCTGGGAATATCCGATGTAGCCTGGTCGTCAGATT CTAACCTTCTTGTTTCTGCCTCAGATGACAAAACCTTGAAGATATGGGACGTG 636399-Uni Würzburg-an-1 GATTGTACAGAAACTACAAGGCCACACAGATGTCGTGATCTCAACAGCTTGTC ACCCAACAGAAAACATCATCGCCTCTGCTGCGCTAGAAAATGACAAAACAATT AAACTGTGGAAGAGTGACTGCACGCGTGGAGTGCAGGTGGAAACCATCTCC CCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCA CTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGGGACAGAA ACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGA AGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCT CCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGC ID NO 29) ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCT ACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAA TCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCG ACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTAC GCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTC GACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTAT CGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATC AACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGGAGCTCC GGTGGGAGCTCCGGTGAATTCGCGACGGAGGAGAAGAAGCCCGAGACCGA 636399-Uni Würzburg-an-1 GAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACC TGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTC CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATC CGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAA CTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATC CCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATGA c. 2xGSSG; NanoLuc-WDR5-FKBP12 NanoLuc-linker- linker(2xGSSG)-FKBP12 (SEQ ID NO 30) ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCT ACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAA TCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCG ACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTAC GCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTC GACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTAT CGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATC 636399-Uni Würzburg-an-1 AACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGGAGCTCC GGTGGGAGCTCCGGTGAATTCGCGACGGAGGAGAAGAAGCCCGAGACCGA GGCCGCCAGAGCACAGCCAACCCCTTCGTCATCCGCCACTCAGAGCAAGCC TACACCTGTGAAGCCAAACTATGCTCTAAAGTTCACCCTTGCTGGCCACACCA AAGCAGTGTCCTCCGTGAAATTCAGCCCGAATGGAGAGTGGCTGGCAAGTTC ATCTGCTGATAAACTTATTAAAATTTGGGGCGCGTATGATGGGAAATTTGAGAA AACCATATCTGGTCACAAGCTGGGAATATCCGATGTAGCCTGGTCGTCAGATT CTAACCTTCTTGTTTCTGCCTCAGATGACAAAACCTTGAAGATATGGGACGTG GGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAG CGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAG AAATTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAA GCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGG TCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGC ACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTA AAACTGGAATGA d. 4xGSSG; NanoLuc-WDR5-FKBP12 NanoLuc-linker-WDR5-linker(4xGSSG)-FKBP12 (SEQ ID NO 31) 636399-Uni Würzburg-an-1 ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCT ACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAA TCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCG ACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTAC GCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTC GACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTAT CGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATC AACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGGAGCTCC GGTGGGAGCTCCGGTGAATTCGCGACGGAGGAGAAGAAGCCCGAGACCGA GGCCGCCAGAGCACAGCCAACCCCTTCGTCATCCGCCACTCAGAGCAAGCC TACACCTGTGAAGCCAAACTATGCTCTAAAGTTCACCCTTGCTGGCCACACCA AAGCAGTGTCCTCCGTGAAATTCAGCCCGAATGGAGAGTGGCTGGCAAGTTC ATCTGCTGATAAACTTATTAAAATTTGGGGCGCGTATGATGGGAAATTTGAGAA AACCATATCTGGTCACAAGCTGGGAATATCCGATGTAGCCTGGTCGTCAGATT CTAACCTTCTTGTTTCTGCCTCAGATGACAAAACCTTGAAGATATGGGACGTG TCTGGCGGAAGCTCTGGCACGCGTGGAGTGCAGGTGGAAACCATCTCCCCA 636399-Uni Würzburg-an-1 GGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTAC ACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGGGACAGAAACAA GCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAA GGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAG ATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACT e. 8xGSSG; NanoLuc-WDR5-FKBP12 NanoLuc-linker-WDR5-linker(8xGSSG)-FKBP12 (SEQ ID NO 32) ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCT ACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAA TCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCG ACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTAC GCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTC GACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTAT CGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATC AACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGGAGCTCC GGTGGGAGCTCCGGTGAATTCGCGACGGAGGAGAAGAAGCCCGAGACCGA GCGTGAGGATATGGGATGTGAAAACAGGGAAGTGCCTCAAGACTTTGCCAGC TCACTCGGATCCAGTCTCGGCCGTTCATTTTAATCGTGATGGATCCTTGATAGT 636399-Uni Würzburg-an-1 TCTGGCGGAAGCTCTGGCGGAAGCTCTGGCGGAAGCTCTGGCGGAAGCTCT GGCGGAAGCTCTGGCACGCGTGGAGTGCAGGTGGAAACCATCTCCCCAGGA GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACC GGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGGGACAGAAACAAGC CCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGG GGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATT ATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCT The NanoLuc-WDR5-FKBP12 construct was generated similar to WDR5-NanoLuc- FKBP12 construct with a 2xGSSG as linker, and the assay was repeated to compare the degradation of both fusion proteins by FBXL12-FRB. The position of the NanoLuc in the WDR5-FKBP12 fusion protein was irrelevant to its activity and deg- radation by FBXL12 as shown in Fig.18. A NanoLuc-WDR5-FKBP12 constructs with 0 to 8x GSSG as linker between WDR5 and FKBP12 was made and the assay was performed with FBXL12-FRB or with FRB. The results from the immunoblot and nanoluciferase assay are shown in Fig. 19 and Fig. 20. Together the results showed both the cellular expression of the WDR5 fusion proteins and their degradation by FBXL12 decreased with an increase of the linker length. Thus, both tight and more flexible arrangements between the target protein and the dimerization tag allow quantification of E3 ligase activity, but a perfect fusion shows the most efficient degradation. 636399-Uni Würzburg-an-1

Claims

Claims 1. System for checking whether a protein of interest (POI) can act as a sub- strate for an enzyme selected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase in a eukaryotic cellular envi- ronment, wherein the system comprises - a first recombinant protein comprising or consisting of an amino acid se- quence of the selected enzyme and an amino acid sequence of a first bind- ing protein of a dimerization system dimerizing the first binding protein with a second binding protein upon addition of a ligand, wherein a first linker is optionally arranged between the amino acid sequence of the selected en- zyme and the amino acid sequence of the first binding protein or - a first recombinant nucleic acid coding for the first recombinant protein, - a second recombinant protein comprising or consisting of - an amino acid sequence of the POI and an amino acid sequence of the second binding protein of the dimerization system, wherein a sec- ond linker is optionally arranged between the amino acid sequence of the POI and the amino acid sequence of the second binding protein, and - an amino acid sequence of a reporter protein or reporter peptide, wherein a third linker is optionally arranged between the amino acid sequence of the POI and the amino acid sequence of the reporter protein or reporter peptide or between the amino acid sequence of the second binding protein and the amino acid sequence of the re- porter protein or reporter peptide, wherein the reporter protein or 636399-Uni Würzburg-an-1reporter peptide is any protein or peptide that can be detected di- rectly or after conversion of a further substrate indirectly by physical means, or - a second recombinant nucleic acid coding for the second recombinant pro- tein and - the ligand, wherein in the second recombinant protein - the reporter protein or reporter peptide and the second binding protein and the second linker and / or third linker in case of presence in the second re- combinant protein are chosen such that no lysine residue is present in the amino acid sequences of the reporter protein or reporter peptide, the sec- ond binding protein and the second linker and / or third linker or - any lysine residue in the amino acid sequence of the reporter protein or re- porter peptide, in the amino acid sequence of the second binding protein and in the amino acid sequence of the second linker and / or third linker in case of presence in the second recombinant protein is replaced by an argi- nine residue or histidine residue.

2. System according to claim 1, wherein the first binding protein of the dimeri- zation system is FKBP-rapamycin binding domain of mTOR complex 1 (FRB) or its 636399-Uni Würzburg-an-1analog FRBT78Lor another analog of FRB and the second binding protein of the di- merization system is 12-kDa FK506 binding protein (FKBP12) or an analog thereof or wherein the first binding protein of the dimerization system is 12-kDa FK506 bind- ing protein (FKBP12) or an analog thereof and the second binding protein of the dimerization system is FKBP-rapamycin binding domain of mTOR complex 1 (FRB) or its analog FRBT78Lor another analog of FRB and wherein the ligand is rapamycin or an analog of rapamycin which analog is able to dimerize FRB or FRBT78Land FKBP12, wherein the analog is in particular AP21967 in case that the first binding protein or the second binding protein is FRBT78L, or in particular temsirolimus, everolimus, ridaforolimus, umirolimus, or zo- tarolimus, wherein any of the before mentioned analogs of FRB and FKBP12 consists of an amino acid sequence having a sequence identity of at least 93 % or a similarity of at least 90 % to the amino acid sequence of FRB and FKBP12, respectively, wherein the sequence similarity is calculated according to the EMBOSS needle al- gorithm having an open gap penalty of 10.0 and an extended gap penalty of 0.5 and using the Blosum 62 matrix.

3. System according to claim 2, wherein the sequence identity of the amino acid sequence of any of the analogs of FRB and FKBP12 to the amino acid se- quence of FRB and FKBP12, respectively, is at least 95 %, in particular at least 97 % and at most less than 100 % and wherein the sequence similarity of the amino acid sequence of any of the analogs of FRB and FKBP12 to the amino acid se- quence of FRB and FKBP12, respectively, is at least 95 %, in particular at least 97 % and at most less than 100 %. 636399-Uni Würzburg-an-14. System according to any of the preceding claims, wherein the amino acid sequence of FRB is the amino acid sequence corresponding to nucleic acid se- quence SEQ ID NO 11 and / or the amino acid sequence of FKBP12 is SEQ ID NO 13.

5. System according to claim 1, wherein the dimerization system is GAI and GID1 system consisting of GAI-tag sequence SEQ ID NO 16, GID1-tag sequence SEQ ID NO 17 and dimerization agent gibberellic acid or gibberellic acid acetoxymethyl ester or Halo-tag and SNAP-Tag system consisting of Halo-tag sequence SEQ ID NO 18, SNAP-Tag sequence SEQ ID NO 19 and dimerization agent HAXS8 or ABI and PYL1 system consisting of ABI-tag sequence SEQ ID NO 20, PYL1-tag sequence SEQ ID NO 21 and dimerizing agent abscisic acid or ABI and PYR1 system consisting of ABI-tag sequence SEQ ID NO 20, PYR1-tag sequence SEQ ID NO 22 and dimerizing agent mandipropamid or DHFR and Halo-tag system consisting of DHFR-tag sequence SEQ ID NO 23, Halo- tag sequence SEQ ID NO 18 and dimerizing agent trimethoprim-HaloTag ligand (TMP-Htag). 636399-Uni Würzburg-an-16. System according to any of the preceding claims, wherein in the second re- combinant protein any lysine residue in the amino acid sequence of the reporter protein or reporter peptide, in the amino acid sequence of the second binding pro- tein, in particular of FKBP12 or the analog thereof or of FRB or of its analog FRBT78Lor of the other analog of FRB, and in the amino acid sequence of the sec- ond linker and / or third linker in case of presence in the second recombinant pro- tein is replaced by an arginine residue or histidine residue, in particular an arginine residue.

7. System according to any of the preceding claims, wherein the system com- prises the first recombinant nucleic acid and the second recombinant nucleic acid and the ligand or the first recombinant protein and the second recombinant protein and the ligand.

8. System according to claim 7, wherein the first recombinant nucleic acid is contained in an expression vector, in particular a plasmid or a virus, or a lentiviral vector and the second recombinant nucleic acid is contained in the same or a fur- ther expression vector, in particular in particular the plasmid or a further plasmid or the virus or a further virus or the lentiviral vector or a further lentiviral vector, or wherein the first and the second recombinant nucleic acid are contained in the ge- nome of an animal cell, in particular a mammalian cell, after introduction by CRISPR knock-in technique.

9. System according to any of the preceding claims, wherein the reporter pro- tein or reporter peptide is a luciferase, in particular firefly-luciferase, 19 kDa cata- lytic subunit of Oplophorus luciferase, in particular a luciferase having sequence SEQ ID NO 1, or a luciferase having sequence SEQ ID NO2 (HiBiT), or a fluoro- phore, in particular green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein(CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), far-red fluorescent protein, infra- red fluorescent protein, mNeonGFP (mNG), enhanced GFP (EGFP) or mCherry. 636399-Uni Würzburg-an-110. System according to any of the preceding claims, wherein the E3-ubiquitin ligase is von Hippel-Lindau E3-ubiquitin ligase (VHL), F-box protein (FBXL12) of SCF (Skp1-CUL-1-F-box protein) type E3 ubiquitin ligase complex or cereblon E3- ubiquitin ligase (CRBN) and / or wherein the POI is oncoprotein Myc, oncoprotein MycN , oncoprotein MycL, aurora kinase A (AURKA), aurora kinase B (AURKB), BCR-ABL fusion protein, signal transducer and activator of transcription 3 (STAT3), GTPase Kras (KRAS), EWS–FLI fusion protein, epidermal growth factor receptor (EGFR), Erb-b2 receptor tyrosine kinase 2 (ERBB2), ALK tyrosine kinase receptor (ALK), Wee1-like protein kinase (WEE1), catenin beta-1 (CTNNB1), Yes- associated protein 1 (YAP1), WD repeat-containing protein 5 (WDR5), RuvB-like AAA ATPase 1 (RUVBL1), RuvB-like AAA ATPase 2 (RUVBL2), transcription elongation factor SPT5 (SUPT5H), transcription elongation factor SPT6 (SUPT6H), transcription elongation factor SPT4 (SUPT4H), transformation / tran- scription domain-associated protein (TRRAP), HECT-UBA and WWE domain con- taining E3 ubiquitin protein ligase 1 (HUWE1), Bruton tyrosine kinase (BTK), inter- leukin 1 receptor associated kinase 4 (IRAK4), interleukin 1 receptor associated kinase 3 (IRAK3), mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), microtubule associated protein tau (MAPT), mutant huntingtin (mHtt), TAR RNA-binding protein (TARDBP), FIS RNA-binding protein (FUS), glycogen synthase kinase 3 beta (GSK3B), or synuclein alpha (SNCA).

11. Use of the system according to any of the preceding claims for checking whether a protein of interest (POI) can act as a substrate for an enzyme selected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a ki- nase or a phosphatase, wherein the first recombinant protein, the second recombi- nant protein and the ligand are brought into contact in eukaryotic cells or an envi- ronment comprising all constituents of an eukaryotic cellular environment or wherein the first recombinant nucleic acid and the second recombinant nucleic acid are co-expressed in eukaryotic cells in cell culture followed by a treatment of the cells with the ligand, wherein a change of cellular concentration of the POI is monitored by measuring the amount or concentration of the reporter protein or 636399-Uni Würzburg-an-1reporter peptide in the eukaryotic cells, in the environment comprising all constitu- ents of an eukaryotic cellular environment or in medium in which the eukaryotic cells are suspended.

12. Use according to claim 11, wherein the reporter protein or reporter peptide is a luciferase, wherein its amount or concentration in the cells, in the environment comprising all constituents of an eukaryotic cellular environment or in the medium is measured by measuring of luminescence after conversion of a further substrate by the luciferase, wherein the further substrate is introduced into the cells or added to the environment comprising all constituents of an eukaryotic cellular environ- ment or wherein the cells are lysed and subsequently mixed with the further sub- strate.

13. Method for checking whether a protein of interest (POI) can act as a sub- strate for an enzyme selected from an E3-ubiquitin ligase, a SUMO E3 ligase, a deubiquitinating enzyme, a kinase or a phosphatase by use of the system accord- ing to any of claims 1 to 10, wherein the method comprises the following steps: - amplifying nucleic acid fragments coding for the selected enzyme, for the first binding protein of the dimerization system dimerizing the first binding protein with the second binding protein upon addition of the ligand, for the second binding protein of the dimerization system, for the reporter protein or reporter peptide, for the POI and optionally for the first, the second and / or the third linker by use of pol- ymerase chain reaction (PCR), wherein the reporter protein or reporter peptide is a further enzyme, wherein in the second recombinant protein - the reporter protein or reporter peptide and the second binding protein and the second linker and / or third linker in case of presence in the second re- combinant protein are chosen such that no lysine residue is present in the amino acid sequences of the reporter protein or reporter peptide, the sec- ond binding protein and the second linker and / or third linker 636399-Uni Würzburg-an-1or - any lysine residue in the amino acid sequence of the reporter protein or re- porter peptide, in the amino acid sequence of the second binding protein and in the amino acid sequence of the second linker and / or third linker in case of presence in the second recombinant protein is replaced by an argi- nine residue or histidine residue, - cloning the amplified nucleic acid fragments into one or more expression vectors such that the resulting nucleic acid or nucleic acids code for the first recombinant protein and the second recombinant protein, - transformation of competent bacteria with the vector(s), selecting and ex- panding bacteria containing the vector(s) and extracting the amplified vec- tor(s), - culturing eukaryotic cells in cell culture, - transfection of the vector(s) into the eukaryotic cells, - expressing the first recombinant protein and the second recombinant pro- tein in the eukaryotic cells, - treating the eukaryotic cells with the ligand, optionally in combination with a cell membrane penetration promoting substance, - lysing the eukaryotic cells, adding a further substrate for conversion by the reporter protein or reporter peptide and (an) additional substance(s) in case the additional substance(s) is / are required for a conversion of the further substrate by the reporter protein or reporter peptide and determining whether a conversion of the further substrate changes over time, wherein a 636399-Uni Würzburg-an-1change of the conversion is an indication for the acting of the POI as a sub- strate for the selected enzyme.

14. Method according to claim 13, wherein the eukaryotic cells are mammalian cells, in particular HEK293 cells and / or wherein the first binding protein of the di- merization system is FKBP-rapamycin binding domain of mTOR complex 1 (FRB) or its analog FRBT78Lor another analog of FRB, the second binding protein of the dimerization system is 12-kDa FK506 binding protein (FKBP12) or an analog thereof and the ligand is rapamycin or an analog of rapamycin able to dimerize FRB or FRBT78Land FKBP12, in particular AP21967 in case that the first binding protein is FRBT78L, temsirolimus, everolimus, ridaforolimus, umirolimus, or zotaroli- mus, wherein any of the before mentioned analogs of FRB and FKBP12 consists of an amino acid sequence having a sequence identity of at least 93 % or a simi- larity of at least 90 % to the amino acid sequence of FRB and FKBP12, respec- tively, wherein the sequence similarity is calculated according to the EMBOSS needle algorithm having an open gap penalty of 10.0, an extended gap penalty of 0.5 and using the Blosum 62 matrix.

15. Method according to claim 13 or 14, wherein the E3-ubiquitin ligase is von Hippel-Lindau E3-ubiquitin ligase (VHL), F-box protein (FBXL12) of SCF (Skp1- CUL-1-F-box protein) type E3 ubiquitin ligase complex or cereblon E3-ubiquitin lig- ase (CRBN) and / or wherein the POI is oncoprotein Myc, oncoprotein MycN , onco- protein MycL, aurora kinase A (AURKA), aurora kinase B (AURKB), BCR-ABL fu- sion protein, signal transducer and activator of transcription 3 (STAT3), GTPase Kras (KRAS), EWS–FLI fusion protein, epidermal growth factor receptor (EGFR), Erb-b2 receptor tyrosine kinase 2 (ERBB2), ALK tyrosine kinase receptor (ALK), Wee1-like protein kinase (WEE1), catenin beta-1 (CTNNB1), Yes-associated pro- tein 1 (YAP1), WD repeat-containing protein 5 (WDR5), RuvB-like AAA ATPase 1 (RUVBL1), RuvB-like AAA ATPase 2 (RUVBL2), transcription elongation factor SPT5 (SUPT5H), transcription elongation factor SPT6 (SUPT6H), transcription elongation factor SPT4 (SUPT4H), transformation / transcription domain-associated protein (TRRAP), HECT-UBA and WWE domain containing E3 ubiquitin protein 636399-Uni Würzburg-an-1ligase 1 (HUWE1), Bruton tyrosine kinase (BTK), interleukin 1 receptor associated kinase 4 (IRAK4), interleukin 1 receptor associated kinase 3 (IRAK3), mitogen-ac- tivated protein kinase kinase kinase kinase 1 (MAP4K1), microtubule associated protein tau (MAPT), mutant huntingtin (mHtt), TAR RNA-binding protein (TARDBP), FIS RNA-binding protein (FUS), glycogen synthase kinase 3 beta (GSK3B), or synuclein alpha (SNCA).

16. Method according to any of claims 13 to 15, wherein the reporter protein or reporter peptide is a luciferase, in particular firefly-luciferase, 19 kDa catalytic sub- unit of Oplophorus luciferase, in particular a luciferase having sequence SEQ ID NO 1, or a luciferase having sequence SEQ ID NO2 (HiBiT), or a fluorophore, in particular green fluorescent protein (GFP), red fluorescent protein (RFP), cyan flu- orescent protein(CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), far-red fluorescent protein, infra-red fluo- rescent protein, mNeonGFP (mNG), enhanced GFP (EGFP) or mCherry.

17. Method according to any of claims 13 to 16, wherein the cell membrane penetration promoting substance is dimethyl sulfoxide (DMSO). 636399-Uni Würzburg-an-1

Images