GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit

CRISPR Human - Deletion FUT8

Experiment
CRISPR Human - Deletion FUT8
Product
GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

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

CRISPR/Cas9-mediated genome editing
To generate FUT8-knockout (FUT8-KO) cells with the CRISPR/Cas9 system, guide RNAs (gRNAs) targeting the human FUT8 gene at exon 3 or 6 were cloned into the GeneArt CRISPR Nuclease Vector (Thermo Fisher Scientific, Waltham, MA, USA). After sequence verification of the insert, the CRISPR/Cas9 plasmids were transfected into HEK-293 T or MDA-MB-231 cells. Two days after transfection, cells underwent flow cytometry-based sorting of crRNA (CRISPR RNA)-expressing cell populations with orange fluorescent protein (OFP) expression. These crRNA-expressing cell populations were further cultured for 1 week, and FUT8-KO cells were selected by fluorescence-activated cell sorting (FACS) analysis with LCA binding. Genomic indel modification of FUT8 in single-cell clones was assessed by PCR and sequencing.

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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 - Deletion FUT8 using GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit from Thermo Fisher Scientific.

Paper title
FUT8 promotes breast cancer cell invasiveness by remodeling TGF-β receptor core fucosylation
Full paper
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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit below.

Download PDF Download manufacturer protocol

Videos

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