Step 1: Construction and testing of CRISPR/Cas9 vectors
This step takes about 2 weeks.
1. Discuss the CRISPR/Cas9 design and targeting strategy with the investigator.
2. Construct the plasmid expressing sgRNA and Cas9 DNA sequencing to confirm each construct.
3. Test the cleavage activity of each sgRNA by co-tranfection with a reporter plasmid in HEK293T cells or in ES cells. Up to three sgRNA expressing plasmids will be tested. The one with the highest cleavage activity will be chosen for gene targeting.