Team:Calgary/Results
RESULTS
Results Overview
The Calgary iGEM team was able to compare the effectiveness of 7 different sgRNA candidates at directing CRISPR/Cas9 cleavage. These results are semi-quantitative, providing the relative rate of indel formation which can be used as a surrogate measure for cleavage activity and NHEJ. The CCR5-17R sgRNA candidate was found to be the most effective, with an indel formation rate of 11.1%. A more thorough explanation can be found on the CRISPR page.
With respect to CRISPR knock-ins, the Calgary iGEM team was unable 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. The attached graphs demonstrate indel activity of the attempted insertion. More information regarding these graphs can be found on the CRISPR page.
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, we found that a small number of cells trasnfected with all components (CRISPR/Cas9 and sgRNA, FLPo-expressing plasmid, and plasmid with hybrid beta-resolvase: BBa_K2605006) 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. A deeper explanation of this result can be found in the Flp-Beta page. 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