lenti dCAS-VP64_Blast

CRISPR Human - Activation interferon-γ promoter

CRISPR Human - Activation interferon-γ promoter
lenti dCAS-VP64_Blast from Addgene

Protocol tips

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and
the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

Plasmid and Vector Constructs
Plasmids encoding NLS-dCas9-VP64 and MS2-p65-HSF1
(plasmid #61425 and #61426), and dCas9-24xGCN4-v4 and scFvGCN4-sfGFP-VP64 expression plasmids were obtained from
Addgene (plasmid #60910 and #60904). The sgRNA expression
plasmid was obtained from Addgene (plasmid #61427), and the
multiple sgRNA clone vector including BsmBI restriction sites
facilitated the insertion of different gRNA target corresponding
oligo DNAs. The lentiCRISPR-V2 plasmid was obtained from
Addgene (plasmid #52961) and the pGL3-basic plasmid was
obtained from Promega. To obtain the interferon-γ promoter
luciferase reporter plasmid, the promoter fragment was amplified
from peripheral blood mononuclear cells using the forward primer
TTCTGGGCTT-3’), followed by purification and digestion by KpnI
and XhoI restriction enzymes and then ligation into the KpnI-XhoI
clone sites of the pGL3-basic plasmid. Interferon-γ promoter
reporter lacking the sgRNA target site (ΔsgRNA9 and sgRNA10)-
luc was constructed using two-step fusion PCR: forward primer
amplify upstream of the 5’-promoter; forward primer (5’-TTA
primer (5’-CCGCTCGAGCGTAGATATTGCAGATACTTCTGGGCTT3’) amplify downstream of the 5’-promoter. Finally, a full-length
interferon-γ lacking the sgRNA9 and sgRNA10 target site fragments
was amplified using a forward primer (5’-CGGGGTACCGGG
ATTACAGGCGTGAGCCACC-3’) and a reverse primer (5’-CCG
fragments amplified in the first step as templates. The purified
interferon-γ promoter (ΔsgRNA9 and sgRNA10) PCR product was
then ligated into the KpnI- and XhoI-digested pGL3-basic plasmid.
All the expression plasmids were confirmed by sequencing

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lenti dCAS-VP64_Blast from Addgene has not yet been reviewed for this experiment

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3 years 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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Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Activation interferon-γ promoter using lenti dCAS-VP64_Blast from Addgene.

Paper title
Specific Expression of Interferon-γ Induced by Synergistic Activation Mediator-Derived Systems Activates Innate Immunity and Inhibits Tumorigenesis.
Full paper
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Manufacturer protocol

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

Download PDF Download manufacturer protocol


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