CRISPR Human - Deletion CIITA

CRISPR Human - Deletion CIITA
pLX-sgRNA from Addgene

Protocol tips

Protocol tips
Do not add antibiotics to the Plasmid Transfection Medium

Publication protocol

CRISPR/Cas9 mutagenesis
The tetracycline-inducible Cas9 lentiviral vector (pCW-Cas9, also produced by Eric Lander & David Sabatini, and available through Addgene as plasmid: #50661) was used to transduce ECFC-derived EC to create stable inducible Cas9 expressing EC. Guide RNA targeting CIITA and CD58 genes were identified using the online optimized software These guides were cloned into pLX-sgRNA vector (produced by Eric Lander & David Sabatini17 and available through Addgene as plasmid: #50662) and transduced into TetOn-Cas9-EC. Loss-of-function was identified by fluorescence-minus-one staining of HLA-DR (in the case of CIITA mutagenesis) and CD58 (in the case of CD58 mutagenesis) and cells were isolated by single-cell FACS and seeded into microwell titer plates containing Y-27632 (Sigma) for clonal expansion and further analysis.

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pLX-sgRNA from Addgene has not yet been reviewed for this experiment

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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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Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Deletion CIITA using pLX-sgRNA from Addgene.

Paper title
Efficient Gene Disruption in Cultured Primary Human Endothelial Cells by CRISPR/Cas9
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Manufacturer protocol

Download the product protocol from Addgene for pLX-sgRNA below.

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