Antibiotic free dmem

HEK293T cells were grown in antibiotic-free DMEM (Life Technologies) with 10% FBS at 37 °C. Cells were transfected at 50-60% cellular confluency with a 4:1 mix of a piggyBac transposon vector coexpressing CFP, a piggyBac transposase vector and either RCas9–UBAP2L or RCas9–4EBP1 (or RCas9 only) using FuGENE HD transfection reagent (Promega). CFP-positive cells (integrants) were collected by fluorescence-activated cell sorting (FACS), expanded and transfected again with a piggyBac transposon vector constitutively expressing RFP, YFP under the control of a Tet-inducible promoter and a gRNA targeting the YFP reporter. RFP-positive cells were collected by FACS and expanded. Cells were induced with doxycycline (10 ng ml⁻¹) for 36 h and quantified by FACS. For each cell, the YFP/RFP fluorescence ratio was quantified as a metric of RCas9–UBAP2L-mediated or RCas9–4EBP1-mediated post-transcriptional regulation of the target transcript, and CFP fluorescence was used to quantify expression levels of RCas9–UBAP2L, RCas9–4EBP1 or RCas9 only.