CRISPR knockout and CRISPR activation plasmid cloning

For knockout genes, LentiCRISPR v2 backbone vector was a gift from Feng Zhang (Addgene plasmid #52961). Cloning was performed as described in the Addgene protocol⁶⁹. Oligo sequences for sgRNAs KO targeting each gene (Non-targeting, WDR59, RICTOR, SAV1, FRMD6) listed in Supplementary Table ². 5 µg LentiCRISPR v2 vector was digested and dephosphorylated by for 30 min at 37 °C. The digested plasmid was then gel-purified by QIAquick Gel Extraction Kit (Qiagen). The pair of oligos for each gene were phosphorylated and annealed using T4 PNK enzyme in a thermocycler by incubating 30 min at 37 °C and 5 min at 95 °C and ramp down to 25 °C. Annealed oligos were diluted at 1:200 and ligated together with digested vector using Quick ligase (NEB) for 20 min at room temperature. For CRISPRa, LentiSAMv2 (Addgene plasmid #75112) and LentiMPH v2 (Addgene plasmid #89308) were kindly provided by Dr. Feng Zhang⁷⁰. The CRISPRa system, also known as CRISPR/dCas9 Synergistic Activation Mediator (SAM) system includes the LentiMPH v2 construct which encodes for the MS2-P65-HSF1 activator helper complex with a 2A hygromycin resistance marker and the LentiSAMv2 empty vector containing a modified Cas9-VP64, MS2 loops at tetraloop and stemloop 2, as well as a blasticidin resistance marker. Golden-Gate sgRNA cloning on LentiSAMv2 empty vector were performed as described in the protocol⁷⁰. Oligo sequences for sgRNAs targeting each gene (SESN3, WDR59, RICTOR, SAV1, and FRMD6) listed in Supplementary Table ². Briefly, oligo primers for each gene were phosphorylated and annealed using T4 PNK enzyme in a thermocycler by incubating 30 min at 37 °C and 5 min at 95 °C and ramp down to 25 °C. Annealed oligos were diluted at 1:10 and ligated together with lentiSAMv2 vector using T7 ligase (Enzymatics) Quick ligase (NEB) for 20 min at room temperature. The cloned vectors were then transformed into Stbl3 bacteria (Invitrogen) and streaking it onto an LB agar plate for ampicillin selection overnight at 33 °C. Plasmid Miniprep was prepared using Qiagen plasmid miniprep kit.