Step2a: CRISPR/Cas9-mediated gene knockout
It takes up to six months to make a human gene knockout cell line.
1. Cell expansion and mycoplasma testing
2. Electroporation of CRISPR guide RNA and Cas9 expressing plasmid into cells
3. PCR screening of the targeted clones followed by DNA sequencing to verify the correct gene targeting
4. Expansion of mutant clones and freezing down (4 vials for each line)