To engineer a lentiviral transfer vector that expresses dCas9:VP64-T2A-Puro (EF1a-NLS-dCas9(N863)-VP64-T2A-Puro-WPRE), dCas9:VP64-T2A-Blast (EF1a-NLS-dCas9(N863)-VP64-T2A-Blast-WPRE) (Addgene, no. 61,425) was digested with BsrGI and EcoRI. T2A-PuroR was amplified from pLV-TetO-hNGN2-P2A-eGFP-T2A-Puro (Addgene, no. 79823). Fragments were then assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB, no. E2621). To engineer a lentiviral transfer vector that expresses dCas9:KRAB-Puro (EF1a-NLS-dCas9(N863)-KRAB-T2A-Puro-WPRE), dCas9:VP64-T2A-Blast (EF1a-NLS-dCas9(N863)-VP64-T2A-Blast-WPRE) (Addgene, no.61425) was first digested with BamHI and BsrGI. KRAB was then amplified from pHAGE-TRE-dCas9:KRAB (Addgene, no. 50917). Fragments were then assembled using NEBuilder HiFi DNA Assembly Master Mix. dCas9:KRAB-Blast was digested with BsrGI and EcoRI, and T2A-PuroR was amplified from pLV-TetO-hNGN2-P2A-eGFP-T2A-Puro (Addgene, no. 79823). Fragments were then assembled using NEBuilder HiFi DNA Assembly Master Mix. To engineer a lentiviral transfer vector that expresses dCas9:VPR-Puro (EF1a-NLS-dCas9(D10A, D839A, H840A, and N863A)-VPR-T2A-Puro-WPRE), dCas9:VPR was first amplified from SP-dCas9-VPR (Addgene, no. 63798), and T2A-PuroR was amplified from pLV-TetO-hNGN2-P2A-eGFP-T2A-Puro (Addgene, no. 79823). dCas9:KRAB-T2A-Puro was digested with BsiWI and EcoRI. Fragments were then assembled using NEBuilder HiFi DNA Assembly Master Mix.
