pLX-sgRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Do not add antibiotics to the Plasmid Transfection Medium

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 http://crispr.mit.edu. 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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Reviews

Discussion

Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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