pcDNA-dCas9-VP64

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

Artificial transcription factor constructs
ZF fused with effector domain VP64. ZF-97 and ZF-126 with VP64 have been described previously [45]. TALE fused with effector domain VP64. TALE-99 and TALE-128 with VP64 were designed to target the same sites as ZF-97 and ZF-126, respectively. Each TALE targets two more nucleotides than its corresponding ZF (a total of 20 nt) in order to start in the nearest thiamine nucleotide. The custom-designed sequence was engineered by Genecopoeia TALE-TF service.

CRISPR/dCas9 fused with effector domains VP64/p300/VPR. All dCas9 constructs used an inactivated form of S. pyogenes Cas9 protein with two mutations in D10A and H840A. pcDNA3.1-dCas9-VP64 and pcDNA3.1-dCas9-p300 (Addgene plasmid # 47107 and 61357, respectively) had a C-terminal fusion of VP64 domain or human p300 HAT core (aa 1048-1664) domain [39], respectively, driven by a CMV promoter, a gift from Charles Gersbach. The pcDNA-dCas9-No Effector is HA tagged at the C-terminus (Addgene plasmid # 47106). pcDNA-dCas9-VPR [40] with VP64, p65 and Rta fused to its C-terminus (Addgene plasmid 63798) was a gift from George Church. All dCas9 vectors used the same set of four sgRNAs based on the pSP-gRNA backbone vector (Addgene plasmid # 47108). Individual guides were designed using the crispr.mit.edu website tool to find target sequences in MASPIN and REPRIMO proximal promoters (Supplementary Figure S1) and cloned into BbsI sites in the sgRNA expression vector, pSP-gRNA backbone, following Gersbach's protocol [24].

Cell lines, cell culture and transfection
The HEK293T human embryonic kidney cell line was obtained from the American Type Culture Collection (ATCC, Manassas). The H157 human lung cancer cell line was kindly provided by Dr. Robert Winn (University of Colorado Health Science Center). Human breast cancer cell lines MCF7 and SUM159 were obtained from the Tissue Culture Facility of the UNC Lineberger Comprehensive Cancer Center (University of North Carolina, Chapel Hill). SUM159 cells were cultured in F12 media supplemented with 5% FBS, 5 μg/mL insulin, 1 μg/mL hydrocortisone. MCF7 cells were cultured in MEM alpha media supplemented with 10% FBS and 1% each of sodium pyruvate, sodium bicarbonate and non-essential amino acids. HEK293T cells were cultured in DMEM media supplemented with 10% FBS. H157 cells were cultured in RPMI 1640 supplemented with 5% FBS. All cells were cultured with 1% penicillin/streptomycin unless stated otherwise.

Transfections were performed in 6-well plates using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. 5 x105 cells were seeded 24 hours prior to transfection in their corresponding media, washed with PBS and cultured in Opti-MEM (750 μL). Each well was transfected using 4.8 μl of Lipofectamine 2000 in 250 μL of Opti-MEM with 2.4 μg of total plasmid DNA, according to the manufacturer's protocol. All transfections had the same total amount of plasmid DNA and pcDNA3.1 empty vector was added to complete the total amount of 2.4 μg per well. Genomic DNA, total RNA and protein were collected 48 hours post-transfection. The transfection efficiency of these combinations in H157 cells was between 40-50%, as assessed by flow cytometry of a control EGFP plasmid.

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Manufacturer protocol

Download the product protocol from Addgene for pcDNA-dCas9-VP64 below.

Download manufacturer protocol

Videos

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