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<div class = "col-lg-12"> | <div class = "col-lg-12"> | ||
<img class = "fullpg" src="https://static.igem.org/mediawiki/2018/e/e0/T--Calgary--GeneInt.png"> | <img class = "fullpg" src="https://static.igem.org/mediawiki/2018/e/e0/T--Calgary--GeneInt.png"> | ||
− | <p class ="caption"> | + | <p class ="caption"> 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. | ||
+ | </p> | ||
</div> | </div> | ||
Revision as of 03:05, 18 October 2018