Phage ef1 dcas9 krab

The plasmid pHAGE-EF1-dCas9-KRAB (Addgene, a kind gift of Scot Wolfe) was digested with BsrGI (New England BioLabs) and the backbone was gel purified. Gibson assembly was used to insert a gBlock (IDT) containing Gibson arms, a Kozak sequence and coding for a truncated version of the NEDD8 protein (NEDD8-T) that lacked the C-terminus diglycine residues (Supplementary Table 3). The resulting plasmid was sequence verified by Sanger sequencing. For lentivirus production, 5 × 10⁶ HEK293T cells were seeded in a T175 tissue culture flask and transfected with the cargo plasmid as well as packaging plasmids psPAX2 (Addgene) and pCMV-VSVG (Addgene) using serum free medium Opti-MEM and transfection reagent Fugene 6 (Promega). Virus containing medium was collected after 48 h, filtered and 40-fold concentrated using the lenti X concentrator (Takara bio). Virus was pelleted by centrifuging at 1500 × g for 45 min at 4 °C and resuspended in sterile DMEM + 1% BSA. The functional titer of the library virus was estimated from the fraction of puromycin resistant cells after transduction with different amounts of virus using serial dilution method. A low MOI of 0.2 was selected for the transduction of NEDD8 KO MDA-MB-231 cells followed by puromycin selection at 2 μg/ml. The expression of NEDD8 was analyzed by western blotting.