pHR-SFFV-KRAB-dCas9-P2A-mCherry

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

Constructs for CRISPRi
We expressed sgRNAs from sgOpti, a modification of pLenti-sgRNA (Addgene
#71409) with the sgRNA scaffold replaced with the sgRNA-(F+E)-combined optimized
scaffold previously described (31). We generated constructs expressing inducible KRABdCas9 by replacing the SFFV promoter with a TRE3G promoter and the P2A-mCherry
cassette with an IRES-GFP or IRES-BFP cassette in pHR-SFFV-KRAB-dCas9-P2AmCherry (Addgene #60954) (10).

CRISPRi line generation
We generated the inducible CRISPRi cells lines by (i) transducing K562 cells with a
construct expressing rtTA linked by IRES to a neomycin resistance cassette expressed
from an EF1α promoter (ClonTech, Mountain View, CA) and selecting with 200 µg/mL
G418 (Thermo Fisher), then (ii) transducing these rtTA-expressing K562 cells with one
of the KRAB-dCas9 constructs described in the section above. We selected for cells
expressing GFP or BFP by fluorescence activated cell sorting (FACS)

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pHR-SFFV-KRAB-dCas9-P2A-mCherry below.

Download manufacturer protocol

Videos

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