I am studying the limits of homologous recombination in Escherichia coli after a cutting event induced by CAS9. Specifically I am looking at both the amount of material that can be spliced in and the number of cut-sites that can be placed in close proximity to one another.
Using two different mating types of yeast, mat a and mat alpha, I am creating a system which will allow me to bring genes from one strain of yeast to the other. I will start with one haploid organism of each mating type, and then I will allow them to mate – this will cause them to become diploid and the small guide RNA molecules from the Mat a strain will lead the CAS9 complex from the Mat alpha strain to a cut site- where they will splice in a specified segment of junk DNA. I will be trying to splice in different lengths of junk DNA in the hope of finding the limit of this type of integration. For the second half of my experiment I will be using CAS9 yet again to cut DNA. The goal here will be to cut the DNA multiple times in a small area and see how well it goes back together.