The gRNAs we used are designed in silico via the CRISPR Design Tool (http://tools.genome-engineering.org). This CRISPR Design Tool is described in a Nature Protocol paper published Dr. Feng Zhang's group in 2013. The title of the Nature protocol paper is Genome engineering using the CRISPR-Cas9 system.
2 days after the CRISPR plasmids transfection, we will treat the cells with puromycin for 1 day to select the transfected cells. Then we will seed the single cell into about 5 96-plates. When the clonies grow up, we will pick the edited clonies by genotyping.
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