Team:Calgary/Gene Integration Diagram
GENE INTEGRATION DIAGRAM
1. CRISPR/Cas9 wild-type binds targeted DNA location according to sgRNA homology 2. Cas9 wild-type cuts DNA in a double stranded fashion 3. The ssDNA insert brought in along with the RNP CRISPR complex is integrated into the genome 4. This integration happens via the non-homologous end joining DNA repair mechanism 5. This “landing pad” containing the FRT site is recognized by FlpO, which recombines with the FRT site present on the plasmid, thus bringing in the entire plasmid into the genome The plasmid contains an inactive puromycin gene as the cargo, this is the case in our assays, for use as a proof of concept 6. Within the genome, two sets of the Six 1, NPS, Six 2 combination are recognized by beta resolvase, excising the contents in-between Result: Desired Product contains the puromycin gene, which is now active. The circularised/excised DNA is discarded, unable to return to the genome.