pLV hUbC-dCas9-T2A-GFP

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate

Publication protocol

Plasmids
The sgRNA plasmid and lentiviral plasmid encoding dCas9 are available on Addgene44 (Addgene #53188 and #53191). The KRAB domain was cloned in-frame with the dCas9 ORF at the C-terminus using NheI sites. For sgRNA screening, the oligonucleotides containing HS2 protospacer sequences were synthesized (IDT-DNA), hybridized, phosphorylated, and inserted into phU6-gRNA plasmids using BbsI sites. The protospacer target sequences for the panel of 21 HS2 enhancer sgRNAs are provided in Supplementary Table 1. U6-sgRNA expression cassettes were transferred in reverse orientation upstream of the hUbC promoter at the PacI sites. For sgRNA and dCas9 transduction experiments, a puromycin resistance cassette was linked to the dCas9 and dCas9-KRAB effectors using a T2A ribosome skipping peptide.

Cell culture
K562 cells and HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cancer Center Facilities. Cell lines were verified via morphological inspection. K562 cells were maintained in RPMI1640 supplemented with 10% FBS and 1% penicillin-streptomycin. HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. All cell lines were cultured at 37C with 5% CO2.

Lentiviral transduction
K562s were transduced with lentivirus to stably express dCas9 or dCas9-KRAB. To produce VSV-G pseudotyped lentivirus, HEK293T cells were plated at a density of 5.1e3 cells/cm2 in high glucose DMEM (GIBCO, #11995) supplemented with 10% FBS and 1% pencillin-streptomycin. The next day after seeding, cells in 10-cm plates were co-transfected with the appropriate dCas9/dCas9-KRAB lentiviral expression plasmid (20 µg), the second-generation packaging plasmid psPAX2 (Addgene #12260, 15 µg), and the envelope plasmid pMD2.G (Addgene #12259, 6 µg) by calcium phosphate precipitation45. After 14–20 hours, transfection medium was exchanged for 10 mL of fresh 293T medium. Conditioned medium containing lentivirus was collected 24 and 48 hours after the first media exchange. Residual producer cells were cleared from the lentiviral supernatant by filtration through 0.45 µm cellulose acetate filters and concentrated 20-fold by centrifugation through a 100 kDa molecular weight cutoff filter (Millipore). Concentrated viral supernatant was snap-frozen in liquid nitrogen and stored at −80 °C for future use. For transduction, concentrated viral supernatant was diluted 1:10 with K562 media. To facilitate transduction, the cationic polymer polybrene was added at a concentration of 4 µg/mL to the viral media. Non-transduced (NT) cells did not receive virus but were treated with polybrene as a control. The day after transduction, the medium was exchanged to remove the virus. For cells that were transduced with lentivirus containing a puromycin resistance gene, 1 µg/ml puromycin was used to initiate selection for transduced cells approximately 96 hours after transduction.



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Manufacturer protocol

Download the product protocol from Addgene for pLV hUbC-dCas9-T2A-GFP below.

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