pHR-SFFV-KRAB-dCas9-P2A-mCherry

CRISPR Human - Repression MYC

Experiment
CRISPR Human - Repression MYC
Product
pHR-SFFV-KRAB-dCas9-P2A-mCherry from Addgene
Manufacturer
Addgene

Protocol tips

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

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Discussion

Discussion

1 year ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Repression MYC using pHR-SFFV-KRAB-dCas9-P2A-mCherry from Addgene.

Paper title
Systematic mapping of functional enhancer-promoter connections with CRISPR interference
Full paper
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Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Human - Repression MYC using pHR-SFFV-KRAB-dCas9-P2A-mCherry from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

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