sgRNAs targeting 606 Ubiquitin E3 ligases, as well as 243 non‐targeting control (NTC) sequences, were designed by Azimuth2 (Doench et al, 2016 (link)) and the top four picks (on‐target scores > 0.6) were chosen per gene, extended by 5 and 3 prime 3Cs homology and obtained as a pool from Twist Bioscience (Dataset EV1 ). The E3‐targeting sgRNA library was made by 3Cs, as described previously (Wegner et al, 2019 (link), 2020 ; Diehl et al, 2021 (link)). In brief, 3Cs‐DNA was generated by mixing phosphorylated pre‐annealed oligonucleotides (sgRNA‐encoding) with ssDNA (library template plasmid). 3Cs‐DNA was purified and desalted using the GeneJET Gel Extraction Kit (Thermo Fisher) and parts of it were analysed by gel electrophoresis alongside the dU‐ssDNA template. The remaining 3Cs‐DNA was electroporated into 10‐beta electrocompetent E. coli (New England Biolabs) by using a Bio‐Rad Gene Pulser and incubated overnight. The final sgRNA library plasmid DNA was purified using the Maxi Plasmid DNA Prep Kit (Qiagen).
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