Team:Calgary/Description
OUR PROJECT
This year, iGEM uCalgary 2018 sought to address some of the key dilemmas affecting targeted gene integration: accuracy of desired insertions, the maximum size of recombinant DNA, and the expression of integrated DNA once it is within a host chromosome. To this extent, uCalgary developed three main systems in order to construct a cell line which could facilitate large, precise gene insertions which were then protected from transcriptional silencing and methylation upon insertion. These three systems were:
- A CRISPR/Cas9 system, to introduce a recombination target site into the host genome
- A FlpO/Beta resolvase system, to swap desired sequences into the genome at the recombination target site and lock them in
- A Chromatin Modifying Elements system, to stop transcriptional silencing and promoter methylation, as well as reduce gene crosstalk
Keep reading below for a breakdown of each of our three project segments!
CRISPR
Inserting a landing pad into the genome to enable recombination
CRISPR/Cas9 is a mechanism which induces targeted breaks into DNA, allowing the insertion of foreign DNA sequences into the break site. This method was selected for its targeted insertion properties 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 was not predicted to cause any errors.
FLP Recombinase-Beta Resolvase
Integrating our desired genes at the landing pad
After CRISPR has placed the FRT site into the genome, recombination can begin. FlpO recombinase is an enzyme which causes the exchange of two pieces of DNA, as long as both contain the same FRT site. Thus, by providing recombinant DNA containing the same FRT sequence as that which was 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 many of the junk 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 to be integrated in.
Chromatin Modifying Elements
Maintenance of integrated genes via minimization of gene silencing and neighbourhood effects
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.
Microfluidics
Another major hurdle that gene therapies have to overcome is the complexities of scaled-out production. To approach this problem, we worked towards developing components of a microfluidic system that could enable large scale, end-to-end manufacture of autologous gene-therapies. Our Droplet Formation Module is designed for high throughput cell encapsulation, and the production of isogenic cell cultures.
Software
Each year, iGEM teams develop software in conjunction with their research. However, it is difficult to efficiently access these tools due to the sheer volume of wiki content. Thus, we created an online database called SARA, the Software Aggregating Research Assistant, which organizes software tools created by iGEM teams and allows for the simplified searching. SARA also provides the opportunity for old software to be updated to stay current, and decreases the likelihood that teams will create redundant software.