COLLABORATIONS

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CRISPR/Cas9 induces targeted breaks into DNA, allowing for the insertion of foreign DNA sequences into the break site. This method was selected for its targeted insertion ability to knock-in a Flp recognition target (FRT) site into the genome, opening the door to recombination in later steps. The FRT site can be thought of as a target, marking out a site in the genome for precision targeting by recombinase in the following stage. While the maximum knock-in size of CRISPR/Cas9 insertion is limited, the small size of our FRT site is not predicted to cause any errors.

PUBLIC ENGAGEMENT

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After CRISPR places the FRT site into the genome, recombination can begin. FlpO recombinase is an enzyme which causes the exchange of two pieces of DNA, provided both contain the same FRT site. Thus, by providing recombinant DNA containing the same FRT site as the one inserted into the genome using CRISPR, FlpO will integrate the recombinant DNA into the genome. Our FlpO recombination system also involves a second recombination protein known as Beta resolvase. Following the initial recombination mediated by FlpO, Beta performs a second recombination which removes the undesirable sequences contained on the recombinant plasmid, as well as its FRT site. Not only does this clean up the final insert, but it prevents the insert from being removed by FlpO down the road. If the CRISPR stage of the project is thought of as placing a target in the genome, the recombinase stage is firing DNA at the target for integration.

SAFETY

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Gene inserts are at risk of being rendered ineffective even after successful integration into the genome, as the spread of heterochromatin and DNA methylation can cause gene silencing. Furthermore, regulatory elements within both the insert and genome near the locus of integration may interact bidirectionally, leading to changes in gene expression known as neighbourhood effects. Chromatin-modifying elements (CMEs) can help to generate an isolated, protected pocket within the genome, thereby assuring stable and sustained expression of integrated genes within eukaryotic systems.

INTEGRATED

Our journey towards a safer gene therapy was shaped in large part by the exploration of the societal context with which our project exists. By considering the project’s applicability to current research, ethical debates, religious views and established public opinions, Snip, Equip, Flip, evolved over time.

Addressing foreseeable societal concerns, our system was designed within an ex vivo, non-viral approach. However, in meeting with the Spiritual Care Advisory Committee at Alberta Health Services it was apparent that we had not considered all concerns. Alterations of the natural order such as enhancement, or cultural eradication were explored. We discovered that within a strictly therapeutic context, Snip, Equip, Flip’s implications as a biotechnology opens the door to a world of poorly defined regulations and safety concerns that we as undergraduate students were not equipped to handle.

Discussions with Dr. Ian Lewis and Dr. Walter Glannon guided our team to consider framing our project within a research context. Within this new framework ethical considerations were in Dr. Glannon’s eyes irrelevant, based on the work we have done and within the constraints we have set upon ourselves. Further, Dr. Lewis saw much more value in our project as a foundational tool to biological advancement than solely set within a therapeutics setting, thus increasing the reach of the project.