The Calgary iGEM team was not able to sequence confirm the targeted insertion of an FRT site using CRISPR/Cas9. However, attempted integration showed significant indel formation, demonstrating that Cas9 was successfully cleaving the targeted DNA. The issue therefore lay with the incorporation of template DNA through homology directed repair.

Figure 11. Complete Transfection: 26.2%

Figure 12. No-FlpO Control: 26.2%

Despite no sequencing data confirming insertion, the amount of DNA sequenced and analyzed did not cover the entire cell population. This leaves the possibility that integration was successful, albeit at extremely low efficiency. To further support this hypothesis, uCalgary found that a small number of transfected cells successfully grew in puromycin-containing cell media. Puromycin resistance was placed promoter-less on a plasmid which was designed to enter the genome through Flp recombinase. Thus, growth in puromycin indicates the possibility that not only did some cells successfully receive an FRT insert from CRISPR, but they also had our desired plasmid integrated into their genome using our recombinase system at the FRT site. We are optimistic about this qualitative measure of success and hope to expand upon these results in the brief time before the Jamboree.

Living HEK cells, adhered to plate

Dead HEK cells, no adherence to plate

Single HEK cell adhering to plate in puromycin antiobiotic, following transfection with all components