pHR-SFFV-dCas9-BFP-KRAB

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

Plasmid design and construction for CRISPRi in human cells
The sequence encoding mammalian codon optimized Streptococcus pyogenes dCas9 (DNA 2.0) was fused with two C-terminal SV40 nuclear localization sequences (NLS) and tagBFP alone or with the KRAB, CSD, WRPW, VP64, or p65AD domains. Using Gibson cloning we cloned these fusion proteins into MSCV-Puro (Clontech) or pHR (Addgene). sgRNAs were expressed using a lentiviral U6 based expression vector derived from pSico which co-expresses mCherry-T2A–Puro from a CMV promoter. The sgRNA expression plasmids were cloned by inserting annealed oligos into the lentiviral U6 based expression vector that was digested by BstXI and XhoI. To detect indel formation in GFP in human cells, we purified genomic DNA from cells expressing dCas9-KRAB and either a negative control sgRNA or sgGFP-NT1 that had been grown in culture for 3 weeks. We PCR amplified the SV40 promoter and GFP, and sequenced the PCR products.



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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pHR-SFFV-dCas9-BFP-KRAB below.

Download manufacturer protocol

Videos

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