The guide RNAs (sgRNAs) targeting the human TSG101, SDCBP/syntenin-1and ATG7 genes were designed using the CRISPick online software (Broad Institute), and subcloned into the pSpCas9(BB)-2A-Puro plasmid (Addgene no. 48139). Forward and reverse oligos for sgRNAs were custom-synthesised (Tsingke Biotech, Beijing, China) and annealed to generate sticky ends. pSpCas9 plasmid was cleaved with BbsI enzyme (#R3539; New England Biolabs, Beijing, China) and ligated with annealed sgRNA using T4 ligase (Takara, Beijing, China).
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