The CRISPR/Cas9 system contains the LV-cas9-puro and LV-sgRNA-EGFP recombinant lentiviruses vectors. LV-cas9-puro carries a puromycin resistance gene and LV-sgRNA-EGFP contains an enhanced green fluorescent protein (EGFP) tag. The three single guide RNAs (sgRNAs) targeting human NCAPD3 were designed and synthesized by Shanghai Genechem. Sequencing was used to validate the sequences of the synthesized sgRNAs. The LV-cas9-puro and LV-sgRNA-EGFP vectors were constructed by Shanghai Genechem.
First, LV-cas9-puro lentiviruses were used to transfect AGS cells. Three days after transfection, a suitable amount of puromycin was used for 3 days of selection to obtain AGS cells with stable Cas9 expression. Following that, the three LV-sgRNA-EGFP lentiviruses were used to transfect Cas9-AGS cells. After 3 days of transfection, an inverted microscope was used to look for green fluorescent protein (GFP), and the percentage of green, fluorescent cells was calculated.
First, LV-cas9-puro lentiviruses were used to transfect AGS cells. Three days after transfection, a suitable amount of puromycin was used for 3 days of selection to obtain AGS cells with stable Cas9 expression. Following that, the three LV-sgRNA-EGFP lentiviruses were used to transfect Cas9-AGS cells. After 3 days of transfection, an inverted microscope was used to look for green fluorescent protein (GFP), and the percentage of green, fluorescent cells was calculated.
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