KRAB-dCas9 Expression in HPSI0214i_kucg-2 Cells

HPSI0214i_kucg-2 cells were engineered to express KRAB-dCas9 from a doxycycline-inducible promoter at the AAVS1 locus^{71 (link)} using pAAVS1-PDi-CRISPRn (a gift from B. Conklin; Addgene, plasmid 73500; http://n2t.net/addgene:73500; RRID: Addgene_73500). The cultures were selected with 100 µg ml⁻¹ G418 until stable colonies originating from single cells formed. The colonies were picked and screened for heterozygous insertion by PCR using two primers flanking the AAVS1 locus (5′-CGAGAGCTCAGCTAGTCTTC-3′ and 5′-CTCTCCCTCCCAGGATCC-3′) and an additional primer binding the insert (5′-GTTCATTCAGGGCACCGGAC-3′). KRAB-dCas9 expression in positive clones was assessed by flow cytometry and immunoblotting after the addition of 2 µM doxycycline. Genome integrity was verified by G-band analysis of expanded clones.