Team:Calgary/Description
OUR PROJECT
This year, iGEM Calgary 2018 sought to address some of the key issues affecting targeted gene integration: accuracy of desired insertions, maximum size of recombinant DNA, and expression of integrated DNA after integration with the host genome. To this extent, Calgary developed three main systems in order to construct a cell line which could facilitate large, precise gene insertions which are protected from transcriptional silencing and methylation. 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 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. Our target insert is called Six/FRT. The Six/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 Six/FRT site is not predicted to cause any errors.
FLP Recombinase-Beta Resolvase
Integrating our desired genes at the landing pad
After CRISPR places the FRT site into the genome, recombination can begin. Recombination is the component of our project which physically inserts desired genes into the genome. While CRISPR can only reliably insert genes of a few kilobases, recombination can insert genes of hundreds of kilobases without issue. FlpO recombinase is an enzyme that causes the exchange of two pieces of DNA that both contain the same FRT site. Recombinant DNA containing the same FRT site as the one inserted into the genome, using CRISPR, will allow FlpO to integrate the recombinant DNA into the genome. A second recombination protein known as Beta resolvase is also used. Following the initial FlpO mediated recombination, Beta performs a second recombination event that removes undesirable sequences on the recombinant plasmid, as well as its FRT site. This clean up of the final insert prevents the insert from being removed by FlpO.
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.
Parts
We designed a toolkit for targeted gene integration and expression maintenance in eukaryotic chassis. Parts include chromatin-modifying elements as well as the mCherry-BGH reporter gene with our improved CMV promoter. The parts have restriction sites for directional cloning into a Multiple Cloning Site (MCS) flanked by distinct FRT (FLP recombinase recognition target) sites.
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.