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The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation.1 Unlike traditional enzyme inhibitors, such molecular glue degraders act sub-stoichiometrically to catalyse rapid depletion of previously inaccessible targets.2 They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Through systematic mining of databases for correlations between the cytotoxicity of 4,518 clinical and pre-clinical small molecules and E3 ligase expression levels across hundreds of human cancer cell lines,3–5 we identified CR8, a cyclin-dependent kinase (CDK) inhibitor,6 as a compound that acts as a molecular glue degrader. A solvent-exposed pyridyl moiety of CR8, in its CDK-bound form, induces CDK12-cyclin K complex formation with DDB1, the CUL4 adaptor protein, bypassing the requirement for a substrate receptor and presenting cyclin K (cycK) for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy to turn target binders into molecular glues.

Molecular glues are a class of small molecule drugs that induce or stabilise protein-protein interactions 1 . In the context of a ubiquitin ligase, drug-induced interactions can lead to protein degradation, which is an emerging strategy for the inactivation of therapeutic targets intractable by conventional pharmacological means 2 . Known molecular glue degraders bind to substrate receptors of E3 ubiquitin ligases and recruit target proteins for their ubiquitination and subsequent degradation by the proteasome.
Thalidomide analogues and aryl sulphonamides are two classes of drugs that act as molecular glue degraders. Widely used in the clinic, thalidomide analogues have proven to be an effective treatment for multiple myeloma, other B cell malignancies, and myelodysplastic syndrome with a deletion in chromosome 5q 7 . Thalidomide analogues recruit zinc-finger transcription factors and other targets to CRBN [8][9][10][11] , the substrate receptor of the cullin-RING E3 ubiquitin ligase CUL4A/B-RBX1-DDB1-CRBN (CRL4 CRBN ) 12 . Similarly, aryl sulphonamides degrade the essential RNA-binding protein RBM39 by engaging DCAF15, the substrate receptor of the CRL4 DCAF15 E3 ubiquitin ligase 13-15 . In these examples, the degraders are not dependent on a ligandable pocket on the target protein, but instead leverage complementary protein-protein interfaces between the receptor and the target. By reprogramming ubiquitin ligase selectivity, these molecules divert the ligase to drive multiple rounds of target ubiquitination in a catalytic manner 16 . Such compounds can thus circumvent limitations of classical inhibitors, expanding the repertoire of "druggable" proteins. Although highly sought-after, molecular glue degraders have only been found serendipitously, and there are currently limited strategies available for identifying or designing such compounds.

CR8 induces proteasomal cycK degradation
To identify small molecules that mediate protein degradation through an E3 ubiquitin ligase, we correlated drug sensitivity data for 4,518 clinical and pre-clinical drugs tested against 578 cancer cell lines 3,4 with the mRNA expression levels for 499 E3 ligase components 5 (Extended Data Fig. 1a). DCAF15 gene expression correlated with indisulam and tasisulam toxicity, consistent with its known function as a degrader of the essential protein RBM39 by the CRL4 DCAF15 E3 ubiquitin ligase, thus demonstrating the potential of the approach (Extended Data Fig. 1b, c). We sought to validate the high-scoring ligase-drug correlations by examining whether CRISPR-mediated inactivation of the identified E3 ligase component would rescue the respective drug-induced toxicity (Extended Data Fig. 1d). These experiments confirmed that sgRNAs targeting DCAF15 confer resistance to indisulam and tasisulam. In addition, we observed a correlation between cytotoxicity of the CDK-inhibitor R-CR8 6 and mRNA expression levels of the CUL4 adaptor DDB1 ( Fig. 1a and Extended Data Fig. 1e). Consistently, sgRNAs targeting DDB1 conferred resistance to R-CR8 (Fig.   1b).
As DDB1-dependent cytotoxicity of R-CR8 implicated ubiquitin ligase-mediated degradation of one or more essential proteins, we performed quantitative proteome-wide mass spectrometry to evaluate protein abundance following compound treatment. Of the >8,000 quantified proteins, cycK was the only protein that consistently showed decreased abundance following R-CR8 addition (Fig. 1c, Extended Data Fig. 1f, g). As expected, R-CR8 did not alter cycK mRNA levels (Extended Data Fig. 1h) and compound-induced cycK degradation could be rescued by inhibition of the E1 ubiquitin-activating enzyme (MLN7243), cullin neddylation (MLN4924) and the proteasome (MG132) (Fig. 1d). Together, these results suggest that R-CR8 triggers rapid proteasomal degradation of cycK ( Fig. 1e) through the activity of a DDB1-containing cullin-RING ubiquitin ligase.
To dissect the molecular machinery required for R-CR8 toxicity, we performed genomewide and E3 ubiquitin ligase-focused CRISPR-Cas9 resistance screens (Fig. 1f, Extended Data Fig. 2a, b). SgRNAs targeting DDB1, CUL4B, RBX1, the cullin-RING activator NEDD8, and the NEDD8-activating enzyme (NAE1/UBA3) were significantly enriched in the R-CR8-resistant cell population. As all of these proteins are required for CRL activity, our results provide genetic evidence for the involvement of a functional CUL4-RBX1-DDB1 ubiquitin ligase complex in mediating R-CR8 cytotoxicity.
Thus far, all known cullin-RING ligases engage their substrates through specific substrate receptors, and DDB1 serves as an adaptor protein able to bind over 20 such receptors (also known as DDB1-CUL4-associated-factors, DCAFs) 17,18 to recruit them to the CUL4-RBX1 ligase core. As no DCAF was identified in our viability screens, we constructed a fluorescent reporter of cycK stability (Extended Data Fig. 2c), in which R-CR8-mediated degradation of endogenous cycK could be recapitulated with a cycK eGFP fusion protein (Fig.  1d, e, Extended Data Fig. 2d-f). Using the stability reporter, in which the extent of degradation can be determined by measuring cycK eGFP levels normalised to mCherry expression, we found that Sand R-CR8 facilitated cycK eGFP degradation to the same extent (Extended Data Fig. 2g; henceforth, CR8 refers to R-CR8). We then performed a genomewide CRISPR-Cas9 screen for genes involved in cycK reporter stability and validated the involvement of DDB1 in CR8-mediated cycK degradation (Fig. 1g, Extended Data Fig. 2h), but not in compound-independent cycK degradation (Extended Data Fig. 2i). In addition, we identified cyclin-dependent kinase 12 (CDK12), which is a known target of CR8 19 and whose activity depends on the interaction with cycK 20 , as a crucial component for CR8induced cycK eGFP destabilisation (Fig. 1g, Extended Data Fig. 2h-k).
As neither the cycK eGFP stability reporter screen nor the CR8 resistance screen identified a substrate receptor, we performed additional CRISPR screens targeting 29 genes encoding known DCAFs or DCAF-like candidate proteins in four different cell lines. While sgRNAs targeting the previously identified components of the CUL4-RBX1-DDB1 complex consistently caused resistance to CR8, a DCAF substrate receptor could not be identified (Extended Data Fig. 3).

CR8 directs CDK12 to CRL4 core component
Since none of our genetic screens highlighted a DCAF required for cycK degradation, we tested whether CR8-engaged CDK12-cycK directly binds one of the CUL4-RBX1-DDB1 ligase components in the absence of a substrate receptor. We therefore performed in vitro coimmunoprecipitation experiments using recombinantly purified proteins. The kinase domain of CDK12 (CDK12 713−1052 ) bound to cycK 1−267 did not markedly enrich DDB1 over the bead binding control in the absence of CR8, whereas equimolar amounts of the compound led to stoichiometric complex formation (Fig. 2a). DDB1 β-propeller domains A (BPA) and C (BPC) 17 , which are otherwise involved in DCAF binding, were sufficient for drug-induced CDK12-cycK recruitment. DDB1 β-propeller B (BPB), which binds CUL4 and is not involved in DCAF binding, was dispensable for the interaction (Fig. 2a). In vitro ubiquitination assays confirmed that the CUL4A-RBX1-DDB1 ligase core alone is sufficient to drive robust cycK ubiquitination (Fig. 2b). Quantification of the interaction showed that CR8 stimulated binding between CDK12-cycK and DDB1 in the range of 100-500 nM depending on the experimental setup (Fig. 2c, Extended Data Fig. 4). While weak CDK12-cycK-DDB1 interaction was still detectable in the absence of the compound in vitro, CR8 strengthened complex formation 500-to 1000-fold as estimated by isothermal titration calorimetry (ITC) (Extended Data Fig. 4f-k). Thus, our data indicate that CR8-engaged CDK12-cycK is recruited to the CUL4-RBX1-DDB1 ligase core through DDB1, and the compound tightens the complex sufficiently to drive CR8-induced cycK degradation in the absence of a canonical DCAF substrate receptor.
We then crystallised CDK12 713−1052 -cycK 1−267 bound to CR8 and DDB1 ΔBPB and determined the 3.5 Å resolution structure of this complex (Fig. 2d, Extended Data Table 1). In the structure, CDK12 forms extensive protein-protein interactions (~2100 Å 2 ) with DDB1. CR8 binds the active site of CDK12 and bridges the CDK12-DDB1 interface, while cycK binds CDK12 on the opposite site and does not contact DDB1. The N-and C-lobes of CDK12 are proximal to DDB1 residues located in a loop of the BPA domain (amino acid (aa) 111-114), BPC-helix 2 (aa 986-990), and a loop in the C-terminal domain (aa 1078-1081) that are otherwise involved in DCAF binding (Extended Data Fig. 5). In addition, the C-terminal extension of CDK12 binds the cleft between the DDB1 domains BPA and BPC, a hallmark binding site of DDB1-DCAF interactions (Extended Data Fig. 5a-d, i). The density for this region could only be tentatively assigned, likely due to the presence of multiple conformations, but the CDK12 C-terminal tail clearly engages with DDB1 and assumes a conformation different from those seen in isolated CDK12-cycK structures (Extended Data Fig. 6a, b, d) 19,21 . Structure-guided mutational analyses combined with time-resolved fluorescence resonance energy transfer (TR-FRET) assays were used to assess the contribution of these interactions to CR8-dependent CDK12-DDB1 complex formation (Fig.  2e, Extended Data Fig. 5e). Taken together, our data demonstrates that CDK12 assumes the role of a glue-induced substrate receptor and places cycK in a position typically occupied by CRL4 substrates (Fig. 2f). This renders CDK12-cycK binding to DDB1 mutually exclusive with that of DCAFs and provides a structural framework for why a canonical substrate receptor is dispensable for cycK ubiquitination.

CDK12-DDB1 interface imparts selectivity
CR8 is a pleiotropic CDK inhibitor reported to bind CDK1/2/3/5/7/9/12 6,19 , yet in cells we observed selective cycK destabilization in the presence of the drug. As cycK is reported to associate with CDK9, CDK12, and CDK13 19 , we tested whether the other cycK-dependent kinases are also recruited to DDB1. The closely related CDK13 (90.8 % sequence identity), but not the more divergent CDK9 (45.5 % sequence identity) (Extended Data Fig. 7a-c), was recruited to DDB1 in the presence of CR8, albeit with a lower binding affinity (Extended Data Fig. 7d-f). Analogously, less productive in vitro cycK ubiquitination was observed for CDK13 compared to CDK12 (Extended Data Fig. 7g). The key difference between CDK9 and CDK12/13 primary sequence lies in the C-terminal extension (Extended Data Fig. 7a, b), which in our structure nestles up against DDB1 BPA and BPC propellers (Fig. 2d, Extended Data Fig. 5i). Mutations in, or truncation of, the CDK12 C-terminal extension (Extended Data Fig. 5c) abolished basal binding between CDK12 and DDB1, whereas complex formation could still be facilitated by CR8 to a varying extent (Extended Data Fig. 7h, i). Hence, our data show that the pan-selective CDK inhibitor CR8 induces specific protein-protein interactions between CDK12/13 and DDB1 and suggest that the Cterminal extension, while contributing to binding, is not essential for drug-dependent kinase recruitment.

CR8 phenylpyridine confers glue activity
CR8 occupies the ATP binding pocket of CDK12 and forms discrete contacts with residues in the BPC domain of DDB1 (~150 Å 2 ) through its hydrophobic phenylpyridine ring system (Fig. 3a, b). Mutation of DDB1 residues Ile909, Arg928, and Arg947 each diminished druginduced recruitment of the kinase (Extended Data Fig. 5f), highlighting the contribution of the phenylpyridine moiety to complex formation. To evaluate the structure-activity relationship underlying the gain-of-function activity of CR8, we probed other CDK inhibitors for their ability to drive complex formation between DDB1 and CDK12. DRF053 22 , a CR8-related inhibitor that carries a differently linked phenylpyridine ring system (Fig. 3a, c), induced binding with two-fold lower affinity than CR8 (Extended Data Fig. 8a). Roscovitine 23 , the parent compound of CR8 that lacks the 2-pyridyl substituent but retains the phenyl ring proximal to Arg928 (Fig. 3a, c), also facilitated complex formation, albeit with a three-fold lower apparent affinity (Extended Data Fig. 8a). The affinity rankorder observed in our TR-FRET assay correlated with the degree of cycK ubiquitination in vitro, in which DRF053 and roscovitine showed less processive ubiquitination (Fig. 3d). As neither DRF053 nor roscovitine induced degradation of the cycK eGFP reporter in cells (Fig.  3e), our results demonstrate that the presence and correct orientation of the 2-pyridyl on the surface of CDK12 confer the gain-of-function activity of CR8 leading to cycK degradation.
To probe whether any ligand could in principle drive the interaction of CDK12 with DDB1, we tested the endogenous CDK nucleotide cofactor ATP for its ability to promote complex formation. ATP neither facilitated nor abrogated the interaction over basal binding observed in the presence of DMSO (Extended Data Fig. 6c), suggesting that although the nucleotidebound conformation of CDK12 seems incompatible with approaching DDB1 (Extended Data Fig. 6b), its C-terminal extension is free to adopt multiple conformations 21 . THZ531 24 , a bulky covalent CDK12/13 inhibitor predicted to clash with DDB1 (Extended Data Fig. 6df), locks the CDK12 C-terminal extension in a conformation incompatible with DDB1 recruitment (Extended Data Fig. 6d). Consistently, THZ531 further decreased the TR-FRET signal and diminished cycK ubiquitination in vitro below DMSO control levels (Fig. 3d, Extended Data Fig. 6c, f) 24 . Flavopiridol 25 , a natural product-derived inhibitor structurally distinct from CR8 (Fig. 3a, c), also stimulated the binding of CDK12-cycK to DDB1 (Extended Data Fig. 8a). While flavopiridol gave rise to moderate cycK ubiquitination in vitro (Fig. 3d), it failed to degrade cycK in cells (Fig. 3e). Our results thus show that the DDB1-compound interactions display significant plasticity and that structurally diverse surface-exposed moieties in CR8, DRF053, roscovitine and flavopiridol can facilitate CDK12-cycK recruitment. Small differences in their ability to stabilise the DDB1-CDK12 complex translate, in an almost binary fashion, into cellular degradation of cycK or lack thereof. This behaviour is reminiscent of CRL4 CRBN and thalidomide analogues 11,26 , where an apparent affinity threshold must be overcome to drive drug-induced target degradation.

CycK degradation adds to CR8 toxicity
Finally, to delineate the contribution of CRL4-mediated cycK degradation to CR8 cytotoxicity over non-degradative CDK inhibition, we compared compound toxicity in wildtype HEK293T Cas9 cells to cells that were pre-treated with MLN4924 (NEDD8-activating enzyme inhibitor), genetically-depleted for DDB1, or subject to DCAF overexpression. Global inhibition of CRL activity by MLN4924 had only minor effects on cell viability (Extended Data Fig. 9a), but resulted in decreased sensitivity to CR8 (Extended Data Fig.  9b), showing that CRL neddylation significantly contributes to CR8 toxicity. Overexpression of the substrate receptor CRBN also affected sensitivity to CR8 and decreased cycK degradation (Extended Data Fig. 9c-g), presumably by reducing the free pool of DDB1. As expected, CR8-induced endogenous cycK degradation was dependent on DDB1 ( Fig. 3f) and, consistently, we found that cytotoxicity of CR8, but not that of the other CDK inhibitors, was ten-fold lower in cells depleted for DDB1 (Fig. 3g, Extended Data Fig. 9h). Together, the data demonstrate that the CRL4-dependent gain-of-function glue degrader activity of CR8 strongly contributes to its cellular potency and provides an additional layer of ortholog-specific CDK inactivation through cycK degradation.
Kinase inhibitors have long been suspected to have a degradation component to their mode of action 27,28 , and our work provides the first characterization and structural dissection of how a kinase inhibitor scaffold acquires degrader properties. Molecular glue degraders have thus far only been shown to engage substrate-recruiting E3 ligase modules. CDK12 is not a constitutive E3 ligase component, but rather serves as a drug-induced substrate receptor, linking DDB1 to the ubiquitination target. CR8 thus bypasses the requirement for a canonical DCAF and instead hijacks the essential adaptor protein DDB1. Although cycK is the primary ubiquitination target, CDK12 may become subject to autoubiquitination upon prolonged compound exposure similar to canonical DCAFs 29,30 .
While previously reported molecular glue degraders engage a ligandable pocket on the ligase to recruit target proteins, CR8 instead binds the active site of CDK12 and does not rely on an independent ligand binding site on DDB1 (Extended Data Fig. 4h). This suggests that the repertoire of targets and ubiquitin ligases accessible to targeted protein degradation can be expanded by target-binding small molecules that induce de novo contacts with a ligase or strengthen existing weak protein-protein interactions. Kinase inhibitors in particular often show poor selectivity and small molecule-induced kinase inactivation that leverages complementary protein-protein interfaces offers a path towards improved drug selectivity, which might, for example, facilitate selective inactivation of CDK12, an emerging therapeutic target 31 .
The gain-of-function glue degrader activity of CR8 is attributed to a 2-pyridyl moiety exposed on the kinase surface. Surface-exposed single residue mutations have been shown to promote the formation of higher-order protein complexes, as the haemoglobin Glu to Val mutation, for example, induces polymerization in sickle cell anaemia 32 . Accordingly, single residue mutations designed to increase surface hydrophobicity give rise to ordered protein assemblies 33 . Bound compounds, such as enzyme inhibitors, can in principle mimic such amino acid changes with dramatic effects on the protein interaction landscape, suggesting that compound-induced protein-protein interactions may be more common than previously recognised. Taken together, our results suggest that modifications of surface-exposed regions in target-bound small molecules offer a rational strategy to develop molecular glue degraders for a given protein target.

Mammalian cell culture
The human HEK293T cell lines were provided by the Genetic Perturbation Platform, Broad Institute and K562 Cas9 , THP1 Cas9 , P31FUJ Cas9 cell lines were provided by Zuzana Tothova

Reporter vectors
The following reporter were used in this study:

Oligos
List of all oligonucleotides used in this study can be found in Supplementary Oligo Table 1.

Bioinformatic screen
We computed Pearson correlations of viability of PRISM repurposing compounds in 8 doses and 578 cell lines 4 with gene expression (GE) and copy number variation (CN) of all detectable protein-coding genes of matched cell lines from The Cancer Cell Line Encyclopedia (CCLE) 5 . Z score was computed for each pair of compounds, dose viability, and genomic feature (GE or CN) across all cell lines. For each compound-genomic feature pair, the most extreme correlations are ranked from negative to positive. To focus on novel compound-gene relationships, we restricted genes to a curated list of 499 E3 ligase components and compounds that are not one of "EGFR inhibitor", "RAF inhibitor" or "MDM inhibitor" based on PRISM repurposing annotation 5 . Hit compounds were selected if either the Z score was less than −6 or ranked in the top 15 with Z score less than −4. The resulting list of 158 E3 gene-compound pairs was further curated and shortened manually to 96 E3 gene-compound pairs, which included 95 unique E3 ligases and 85 unique compounds. . Suspension cells were directly subjected to analysis. 10 μL of cell suspension was subjected to the flow analysis with FACSCanto equipped with High Throughput Sampler (BD Bioscience). The percentage of sgRNA transduced cells in the drug treatment wells was normalised to the DMSO control. Wells with fluorescent drug and samples with less than 120 viable cells events or less than 6% fluorescent cells were removed from analysis. All E3-drug pairs were ranked based on the number of experimental conditions (cell line and drug dose) with more than 50% of sgRNA transduced cells in drug treatment wells in comparison to corresponding DMSO control.

Validation of DDB1 resistance phenotype
For validation experiments, virus was produced in a 6-well plate format, as described above with the following adjustments: 2.5 × 10 5 HEK293T cells/well in 2 mL DMEM medium, 3 μL/well of TransIT-LT1, 15 μL/well of OPTI-MEM, 500 ng/well of the desired plasmid, 500 ng/well psPAX2, and 50 ng/well pVSV-G in 32.5 μL/well OPTI-MEM. After collecting the virus, 10 × 10 3 HEK293T Cas9 cells in 100 μL DMEM medium were transduced with 10 μL of virus supernatant. The transduced HEK293T Cas9 cells were then mixed with untransduced control cells at a 1:9 ratio. Nine days after sgRNA transduction, cells were treated for 3 days with DMSO or 1 μM CR8 and analysed by flow cytometry to determine the percentage of BFP + cells. sgRNAs targeting DDB1 provide partial depletion of DDB1 (50% DDB1 alleles modified, reducing DDB1 levels by roughly 50%), which suggests selection towards heterozygous or hypomorphic clones.
Whole proteome quantification using tandem mass tag mass spectrometry 10 × 10 6 Molt-4 cells were treated with DMSO (triplicate) or 1 μM CR8 (single replicate) for 1 or 5 hours and later were harvested by centrifugation. Samples were processed, measured and analysed as described before 37 . Data are available in the PRIDE repository (PXD016187 and PXD016188).

Quantitative PCR
HEK293T Cas9 cells were treated with DMSO or 1 μM CR8 for 2 hours, collected by centrifugation, washed with PBS, and snap-frozen at −80°C. mRNA was isolated using a QIAGEN RNA kit (Qiagen, 74106). For cDNA synthesis, total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer) before qPCR analysis with TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, 4444557) for CCNK (TaqMan, Hs00171095_m1, Life Technologies) and GAPDH (TaqMan, Hs02758991_g1). Reactions were run and analysed on a CFX96 Real Time system (Bio-Rad).

Immunoblots for whole protein lysate
Cells were washed with phosphate buffered saline (PBS) and lysed (150 mM NaCl, 50 mM Tris (pH 7.5), 1% NP-40, 1% glycerol, 1x Halt Cocktail protease and phosphatase inhibitors) for 20 minutes on ice. The insoluble fraction was removed by centrifugation, protein concentration was quantified using a BCA protein assay kit (Pierce), and equal amount of lysate was run on SDS-PAGE 4-12% Bis-Tris Protein Gels (NuPAGE, Thermo Fisher) and subsequently transferred to nitrocellulose membrane with Trans-Blot Turbo System (BIO-RAD). Membranes were blocked in Odyssey Blocking Buffer/PBS (LI-COR Biosciences) and incubated with primary antibodies overnight at 4°C. The membranes were then washed in TBS-T, incubated for 1 hour with secondary IRDye-conjugated antibodies (LI-COR Biosciences), and washed three times in TBS-T for 5 minutes prior to Near-Infrared Western blot detection on an Odyssey Imaging System (LI-COR Biosciences).

CycK stability reporter analysis
HEK293T Cas9 expressing the cycK eGFP degradation reporter were transduced with experimental sgRNAs. Nine days after infection the cells were dosed for 2 hours with DMSO or 1 μM CR8 and fluorescent signal quantified by flow cytometry (CytoFLEX, Beckman or LSR Fortessa flow cytometer BD Biosciences). Using FlowJo (flow cytometry analysis software, BD), the geometric mean of eGFP and mCherry fluorescent signal for round and mCherry positive cells was calculated. The ratio of eGFP to mCherry was normalised to the average of three DMSO-treated controls.  (Fig. 1f) 38 .

BISON CRISPR -CR8 resistance screen
The BISON CRISPR library targets 713 E1, E2, and E3 ubiquitin ligases, deubiquitinases, and control genes and contains 2,852 guide RNAs. It was cloned into the pXPR003 as previously described 35 by the Broad Institute Genome Perturbation Platform (GPP). The virus for the library was produced in a T-175 flask format, as described above with the following adjustments: 1.8 × 10 7 HEK293T cells in 25 mL complete DMEM medium, 244 μL of TransIT-LT1, 5 mL of OPTI-MEM, 32 μg of library, 40 μg psPAX2, and 4 μg pVSV-G in 1 mL OPTI-MEM. 10% (v/v) of BISON CRISPR library was added to 6 × 10 6 HEK293T Cas9 cells in triplicates and transduced. Samples (n=3) were processed as describe above for the genome wide resistance screen.

Genome wide CRISPR -cycK stability reporter screen
A single clone of cycK eGFP HEK293T Cas9 was transduced with the genome wide Brunello library as described above with the following modification: 4.5 × 10 8 cycK eGFP HEK293T Cas9 cells in 225 mL of medium. Nine days later cells were treated with CR8 (n=3) or DMSO (n=3) for at least 2 hours and the cycK stable population was separated using fluorescence activated cell sorting. Four populations were collected (top 5%, top 5-15%, lowest 5-15% and lowest 5%) based on the cycK eGFP to mCherry mean fluorescent intensity (MFI) ratio on MA900 Cell Sorter (Sony). Sorted cells were harvested by centrifugation and subjected to direct lysis as described above. The screen was analysed as described below by comparing stable populations (top 5% eGFP/mCherry expression) to unstable populations (lowest 15% eGFP/mCherry expression). Hits enriched in cycK stable population with FDR < 0.05 are labelled on the plot (Fig. 1g).

Pooled CRISPR screen data analysis
The data analysis pipeline comprised the following steps: (1) Each sample was normalised to the total number of reads.
(2) For each guide, the ratio of reads in the stable vs. unstable sorted gate was calculated, and sgRNAs were ranked. (3) The ranks for each guide were summed for all replicates. (3) The gene rank was determined as the median rank of the four guides targeting it. (4) P-values were calculated by simulating a distribution with guide RNAs that had randomly assigned ranks over 100 iterations. R scripts can be found in the Supplementary Information.

Co-immunoprecipitation assay
The purified His 6 -CDK12/StrepII-cycK complex was mixed with equimolar concentrations of full-length His 6  Plates were incubated at 19°C and crystals appeared 5-13 days after setup. Crystals were flash cooled in liquid nitrogen in reservoir solution supplemented with 25% (v/v) glycerol as a cryoprotectant prior to data collection. Diffraction data were collected at the Swiss Light Source (beamline PXI) with an Eiger 16M detector (Dectris) at a wavelength of 1 Å and a crystal cooled to 100 K. Data were processed with DIALS, scaled with AIMLESS supported by other programs of the CCP4 suite 41 , and converted to structure factor amplitudes with STARANISO 42 , applying a locally weighted CC 1/2 = 0.3 resolution cutoff.

Structure determination and model building
The DDB1 ΔBPB -R-CR8-CDK12 713−1052 -cycK 1−267 complex formed crystals belonging space group P3 1 21, with three complexes in the crystallographic asymmetric unit (ASU). Their structure was determined using molecular replacement (MR) in PHASER 43 with a search model derived from PDB entry 6H0F for DDB1 ΔBPB , and PDB entry 4NST for CDK12-cycK. The initial model was improved by iterative cycles of building with COOT 44 , and refinement using phenix.refine 45 or autoBUSTER 46 , with ligand restraints generated using eLBOW through phenix.ready_set 47 . The final model was produced by refinement with autoBUSTER. Analysis with MOLPROBITY 48 . indicates that 93.9% of the residues in final model are in favourable regions of the Ramachandran plot, with 0.6% outliers. Data processing and refinement statistics are in Extended Data Table 1. Interface analysis was performed using PISA 49 .

Biotinylation of DDB1
Purified full-length StrepII-Avi-DDB1 was biotinylated in vitro at a concentration of 8 μM
Counter-titrations with unlabelled proteins were carried out by mixing 500 μM Alexa488 cycK-CDK12 with 50 nM biotin DDB1 in the presence of 4 nM terbium-coupled streptavidin and 1 μM compound for DDB1 titrations or 12.5 μM compound for CDK12 titrations. After incubation for 15 minutes at room temperature, increasing amounts of unlabelled cycK-CDK12 or DDB1 (0-10 μM) were added to the preassembled Alexa488 cycK-CDK12/ biotin DDB1 complexes in a 1:1 volume ratio and incubated for 15 minutes at room temperature. TR-FRET data were acquired as described above. The 520/490 nm ratios were plotted to calculate the half maximal inhibitory concentrations (IC 50 ) assuming a single binding site using Prism 7 (GraphPad). IC 50 values were converted to the respective K i values as described previously 50 . Three technical replicates were carried out per experiment.

In-vitro ubiquitination assays
In rpm. An initial injection of the ligand (4 μL) was made and discarded during data analysis. For probing DDB1-CDK12-cycK complex formation, DDB1 ΔBPB (100 μM, in the syringe) was titrated into the cell containing CDK12-cycK (10 μM) or CDK12-cycK (10 μM) pre-incubated with CR8 (30 μM). The heat change accompanying the titration was recorded as differential power by the instrument and determined by integration of the peak obtained. Titrations of ligand to buffer only and buffer into protein were performed to allow baseline corrections. The heat change was fitted using nonlinear least-squares minimization to obtain the dissociation constants, K d , the enthalpy of binding, ΔH, and the stoichiometry, n. Between one and three replicates were performed per titration.

Bioluminescence Resonance Energy Transfer (BRET) analyses
Bioluminescence resonance energy transfer (BRET) experiments were using a NanoBRET PPI starter kit (Promega N1821) according to the manufacturer's instructions and as previously described 51 .

CycK stability reporter analysis with CRBN overexpression
HEK293T Cas9 cells expressing the cycK eGFP degradation reporter were transiently transfected with pLX307-Luc or pLX307-CRBN (for flow experiment) as described above and 48 hours post infection treated with CR8 for 2 hours and analysed by flow cytometry.