CRISPR Stable Knockin Cell Line Generation

Using CRISPR to Knock-in Red Fluorescent Protein (RFP) gene into Human Embryonic Kidney Cells at the AAVS1 Safe Harbour Site

• After these passages puromycin was added to the media to select for cells with successful knock-in of the RFP-puromycin resistance CAS:sette.
• After 3-4 weeks of selection, >95% of HEK293 cells were expressing RFP.

Figure 3. After transfection, HEK293 cells were passaged ten times to dilute out the episomal vector, then grown in the presence of puromycin for 4 weeks. Top Left) Cells transfected with both pCAS:-Guide-AAVS1 and pAAVS1-RFP-DNR were healthy after 4 weeks. Top Right) Over 95% of these cells imaged expressed RFP. Bottom Left) Control cells not transfected with the vectors died after puromycin treatment.

• To confirm knock-in of RFP in the genomic DNA, a primer pair was designed with Primer 1 targeting the 5’ homology arm upstream of RFP and Primer 2 targeting within the RFP-Puromycin resistance CAS:sette.
• PCR product of 1.1 kb indicates successful knock-in at AAVS1 site; absence of PCR amplification indicates unsuccessful CAS:sette insertion (Figure 4).
• No PCR amplification was seen in the control cells (‘WT cell’) since Primer 2 could not anneal to the genomic DNA.

Figure 4. Genomic PCR was used to confirm the knock-in of RFP. In edited cells, both primer 1 and primer 2 can bind, resulting in a 1.1 kb PCR product. No PCR product is formed in WT cells as primer 2 cannot anneal to the genomic DNA.

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