Generation of CRISPR Knockout Cell Lines

The pLentiCRISPRv2-LoxP plasmid was generated by inserting LoxP sites (5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′) flankingthe elongation factor-1 α short promoter of plentiCRISPRv2 (Addgene 52961; Shalem et al., 2014 (link)). gRNA sequences targeting lyspersin (5′-GCCGCAGCTTGACGTCTTCG-3′) and LAMTOR1 (5′-GAGGAGGGACGGCGTCCGTG-3′) were chosen using an online prediction tool (CRISPR Design; Zhang laboratory, MIT; Hsu et al., 2013 (link)) and subcloned into pLentiCRISPRv2-LoxP. HEK293LTV were cotransfected with the lentiviral plasmids pVSV-G (631530; Clontech) and psPAX2 (Vogel et al., 2015 (link)) and the viral supernatant directly used to infect HeLa cells. Transfected cells were subsequently selected with puromycin (P7255; Sigma-Aldrich). After selection, cells were transiently transfected with pCAG-Cre:GFP (13776; Addgene; Matsuda and Cepko, 2007 (link)) to remove the Cas9 and FACS sorted to enrich for the GFP-positive population. Limiting dilution then generated single cell clones. Finally, the KOs of lyspersin and LAMTOR1 (also referred to as LAMTOR1HM; de Araujo et al., 2017 (link)) were confirmed by PCR screen (Yu et al., 2014 (link)), Western blot analysis, and genotyping of the respective locus.