The Pathways Research Explorers Program taught me a wide array of laboratory skills and gave me a unique insight into biomedical research and the work being done at Fred Hutch. Many things we learnt and did in this program intrigued me, but using CRISPR/Cas9 to genetically edit a BRCA1 gene was my favorite experience.
Cas9 is a DNA-cutting protein. In CRISPR, a single guide RNA is bound to the protein, forming a Cas9 Complex. The Cas9 Complex can identify and cut specific DNA. Cas9 then locates the section of DNA to edit by looking for a PAM, a short and unique base pair sequence following the targeted DNA sequence to let Cas9 know its in the correct location. Once located, the guide RNA unwinds part of the helix, and binds to the base pair since it has the corresponding nitrogenous bases. Cas9 then cuts that section out of the DNA. The cell may try to repair this break, but usually will make an error, resulting in the disability of the gene, so a specific protein will not be coded for. This makes CRISPR an efficient method for getting rid of certain genes.
In this lab, we chose two guide RNAs of different fragment sizes. We added them into a solution with Cas9 and PCR-grade water, and later added the BRCA1 solution from a previous experiment. After incubating, we added a dye to the solution and performed gel electrophoresis on them to see if their base pair number changed in comparison to uncut BRCA1, which has 1106 base pairs. In conclusion, the two modified DNAs got cut in the areas we thought they would.
Before this program, I had only heard and understood very little about gene editing. I had always perceived it to be an extremely complex task only done by top scientists with years of experience. I was proven wrong as the SEP teachers guided us through the steps until we thoroughly understood what we would be doing in the lab. Once in the lab, my partner and I were able to replicate a process I knew close to nothing about just a couple days earlier. Even though this genetic editing of the BRCA1 gene ended up being fairly straightforward, I felt more confident in my ablilities and potential as a scientist and student after conducting this process.
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