lenti dCAS-VP64_Blast

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

CRISPR dCas9 VP64 promoter activation
Specific guides were designed to target the first 200 bp upstream of the transcription start site (TSS) for both PDIA1 and RhoGDIα (see map at Fig. 6D and E) using a CRISPR designing tool (http://sam.genome-engineering.org/). A scrambled sequence was also designed as control. Sequences were as follows: PDIA1seq. 1-GACAGCGAGCGCGAGGTCCT; PDA1seq. 2-CTTGTCGGGAGCCAATGAG; PDIA1seq. 3-TCCGAATCCGGGGCCAGGCC; RhoGDIαseq. 1-GCCCTGCCTGTCACTTCCG; RhoGDIαseq.2-GCACGCCGTCGCCATCTTG; RhoGDIαseq. 3-GAAGGCCAGGGCGCATGCG; Scrb- GCACTACCAGAGCTAACTCA. Guide oligos were cloned into lenti sgRNA(MS2)_zeo plasmid (a gift from Feng Zhang, Addgene plasmid #61427) according to the Zhang lab SAM cloning protocol available on Addgene (https://www.addgene.org/crispr/zhang/#sam). A nuclease-dead Cas9 construct (lenti dCas-VP64_Blast Addgene plasmid #61425) and a helper complex (lenti MS2-P65-HSF1_Hygro Addgene plasmid #61426) complete this previously described three-vector CRISPR system91. For lentivirus production, lenti sgRNA(MS2)_zeo constructs, lenti dCAS-VP64_Blast and lenti MS2-p65-HSF1_hygro were individually transfected into HEK293T cells using lipofectamine 3000 reagent according to the manufacturer’s protocols. psPAX2 (a gift from Didier Trono, Addgene plasmid # 12260), and pCMV-VSV-G (a gift from Bob Weinberg, Addgene plasmid # 8454) were co-transfected with each plasmid above for effective viral particle production; 48 h after transfection, viral supernatants were collected, filtered, and applied to HUVEC after addition of 8 µg/ml of polybrene or frozen at -80 °C for posterior use. We initially transduced HUVEC with dCAS-VP64_Blast and MS2-p65-HSF1_hygro lentiviral particles. After concomitant selection with both antibiotics, we transduced these cells with sgRNA(MS2)_zeo lentiviral particles carrying each of the specific guide sequences and further selected with Zeocyn. The resultant sublines were designated: PDA1 or RhoGDIα gRNA#1, gRNA#2 and gRNA#3.

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Manufacturer protocol

Download the product protocol from Addgene for lenti dCAS-VP64_Blast below.

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