In CRISPR/Cas9 system, there are two critical parts: sgRNA sequence and Cas9 sequence. These two sequence could be cloned into separated plasmids or combined into one plasmids, each format has its own set of advantages and they share the similar delivery procedure as we describe below.
Two systems cartoons below:
1. Approximately 18-24 hours before transfection, plate cells in 2.5 ml complete growth medium per well in a 6-well plate.
For adherent cells: Plate cells at a density of 0.8 - 3.0 × 105 cells/ml.
For suspension cells: Plate cells at a density of 2.5 - 5.0 × 105 cells/ml.
2. Incubate cell cultures overnight.
Tips: Cell confluency, reagent volume, and post-transfection incubation time are a few key parameters that affect the outcome of transfection experiments.
Do use healthy and actively dividing cells at a confluency 50%-70%.
Do use high quality DNA for transfection, commonly the A260/280 should higher than 1.8.
Do not mix DNA and transfection reagent for too long time.
1. Ensure cells at 50%-70% confluence
2. Prepare DNA-lipid mixture
3. Add DNA-lipid mixture into cells
Generally, Cas9 and sgRNA are detectable as early as 4 hours post-transfection and persisting for many days. With the guidance of sgRNA, Cas9 would cleave target sequence and cause mutations which could be detected by T7E1 assay. The time point for Gene disruption detection can be determined by varying post-transfection incubation times from 4 to 72 hours.