Pu6 sp pegrna hek3 ctt ins

Plasmids pCMV-PE2 (Addgene #132775), pU6-pegRNA-GG-acceptor (Addgene #132777) and pU6-Sp-pegRNA-HEK3_CTT_ins (Addgene #132778) were a gift from David Liu. pMD2.G and psPAX2 (second-generation lenti-viral packaging construct, Addgene #12259, #12260) were a gift from Didier Trono. pLenti_PE2_P2A_Puro vector was constructed by assembling amplified PE2 (from Addgene #132775; primers listed in Table) into BamH1 and Nhe1 digested pLenti-ABERA-P2A-Puro vector (Addgene#112675; a gift from Lucas Dow) using NEBuilder HiFi DNA Assembly Master Mix as per the manufacturer’s instructions. Lenti_pegRNA_GFP/RFP vector was constructed by removing the gRNA scaffold from pLKO5.sgRNA.EFS.GFP/RFP (Addgene #57822/57823; Gift from Benjamin Ebert).

To generate pGL-FL-a or pGL-FL-b plasmids, the binding sequence of siRNAs targeting gene A and gene B was inserted in the 3′-UTR of Firefly luciferase in pGL plasmid. shRNA plasmids were constructed using pU6-Sp-pegRNA-HEK3_CTT_ins (Addgene plasmid # 132778) as a backbone. HEK293T cells in 96-well plates were transfected with 150 ng pGL-FL plasmid, 25 ng of Renilla luciferase (RL) plasmid (pGL-RL), and varying amounts of shRNA plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). After 24 h, the transfected cells were lysed in 20 μL of Promega E1980 lysis buffer, and Firefly and Renilla luciferase activities were measured using luciferase substrate and a multi-mode reader (flexstation 3 multi-mode microplate reader). The relative expression of Firefly luciferase was normalized to that of Renilla luciferase. Refer to Supplementary Table S8 for the sequences of oligos used in this section.