Paavs1 ndi crispri

The Gen1 (pAAVS1-NDi-CRISPRi, a gift from Bruce Conklin, Addgene plasmid # 73497) and pX459 (pSpCas9(BB)-2A-Puro, a gift from Feng Zhang, Addgene plasmid # 48139) containing sgRNA targeting AAVS1 locus were electroporated into HUES8 cells using Nucleofector (Lonza) and cells were selected with 1 μg/μl G418. After 14 days of selection, clonal lines were generated by dilution in 96-well plates, and further selected based on doxycycline induction of mCherry expression.
For the repression of MER57E3 activity, sgRNA was designed by CRISPOR (http://crispor.tefor.net/), and cloned to the LentiGuide-Puro vector (a gift from Feng Zhang, Addgene plasmid # 52963). As for LTR5_Hs/LTR5, 6 sgRNAs were selected from previous reports, and sgRNA pool was cloned to the LentiGuide-Puro plasmid backbone. All sgRNA sequences were provided in Supplementary Table S5. The above plasmids were co-transfected into HEK293T cells together with psPAX2 (a gift from Didier Trono, Addgene plasmid # 12260), pMD2.G (a gift from Didier Trono, Addgene plasmid # 12259) to generate lentivirus. Concentrated lentiviral particles were added to the culture medium of KRAB-dCas9 HUES8 line. After 4 days of selection with 2 ug/ml puromycin, stable cell line was established.