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.

關鍵字  CRISPR Stable Knockin Cell Line Generation；CRISPR Stable Knockin Cell Line Generation；CRISPR Stable Knockin Cell Line Generation；CRISPR Stable Knockin Cell Line Generation；CRISPR Stable Knockin Cell Line GenerationCRISPR Stable Knockin Cell Line Generation

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