To identify the E3 ligase targeted by the degraders, we performed a fluorescence activated cell sorting (FACS)-based CRISPR/Cas9 screen (Fig. ). To this end, we engineered a fluorescent BRD4 protein stability reporter by fusing BRD4S to TagBFP, followed by a P2A self-cleaving peptide and mCherry for normalization. We stably expressed this reporter in KBM7 cells harboring a doxycycline inducible Cas9 allele and then transduced BRD4 reporter cells with a CRL-focused sgRNA library targeting 495 genes with 8 sgRNAs per gene. After selection of library expressing cells, Cas9 expression was induced with doxycycline for 72h, followed by treatment with compounds 9 and 10 or the established BRD4 PROTAC dBET6 . Cells showing elevated (BRD4 HIGH ) or decreased (BRD4 LOW ) BFP levels compared to the bulk population (BRD4 MID ) were isolated via FACS and the sgRNAs enriched in the respective populations were quantified via deep-sequencing (Fig. ). As expected, the dBET6 control screen identified the members of the CUL4 CRBN E3 ligase complex, including the scaffolding protein CUL4A, the E2-adaptor ring box protein RBX1, the DNA damage binding protein DDB1 and the substrate receptor CRBN as top hits (Supplementary Fig. and Supplementary Tables and). Further hits included 20S proteasome subunits and general regulators of CRL activity, such as the COP9 signalosome and the NEDD8-activating E1 enzyme subunit UBA3, thus confirming the successful identification of genes required for BRD4 degradation via dBET6 in the FACS-based CRISPR screen (Fig. and Supplementary Fig. ). For compounds 9 and 10, similar genes were discovered, however, DCAF11 rather than CRBN scored as the most significant CRL substrate receptor in both screens (Fig. , Supplementary Fig. and Supplementary Tables and). These data suggest that bifunctional compounds 9 and 10 recruit the CRL4 DCAF11 complex to induce proteasomal degradation of BRD4 (Fig. ). For target validation, DCAF11 was depleted by RNA interference (Fig. ), which abolished BRD2 degradation induced by 9 (Fig. ). In addition, two different DCAF11targeting sgRNAs were used to generate stable knockouts in KBM7 cells (KO1 and KO2, Fig. ). In both cases, depletion of DCAF11 abolished the BRD2 degradation induced by compounds 9, 10, and 11 (Fig. ). Similar results were obtained for BRD3 and BRD4 (Supplementary Fig. ) and PDE degradation by 5 (Supplementary Fig. ). In addition, treatment of Hep G2 cells with chloroacetamide 14, which was recently identified as DCAF11 binder , partially blocked the degradation of BRD2 by bifunctional degrader 9 (Fig. ). After knockout of the DDB1 adaptor protein by Doxinducible CRISPR/Cas9 expression in KBM7 cells (Supplementary Fig. ) treatment with degraders 9 and 10 did not lead to BRD2 degradation anymore (Fig. and Supplementary Fig. ), and degradation of PDE by treatment with 5 was also abolished (Supplementary Fig. ). After siRNA-mediated depletion of the RING box protein RBX1, compound 9 did not induce degradation of BRD2 anymore (Fig. ).