CRISPR-Cas9 Activity Questions:

1. CRISPR-Cas9 is a biotechnology tool that can be used to edit DNA in cells cheaply and quicky. This technology is a large advancement because it can help prevent harmful mutations developed during DNA replication. These negative mutations are carried in DNA’s genes, which CRISPR- Cas9 is able to inactivate and edit. In class, we explored the capabilities of CRISPR-Cas9 through paper models and a digital model.
2. CRISPR-Cas9 stands for “clustered regularly interspaced short palindromic repeats” which are repeating sequences found in the DNA genome of bacteria. Cas9 stands for “CRISPR-associated protein 9”, which is an enzyme that cuts nucleic acids. The structure of CRISPR-Cas9 has two parts, Guide RNA and the Cas9 Protein. The guide RNA is synthesized to match a certain target sequence, such as a sequence within a gene. The Cas9 protein is an enzyme called a nuclease that is made to cut nucleic acids, and can also break H-bonds. The guide RNA and the Cas9 protein are attached together, like a packaged deal. Once introduced to a human cell, the guide RNA randomly associates with a selected piece of targeted DNA, bringing the Cas9 protein with it. Then, the Cas9 will recognize and bind to a three-nucleotide sequence motif called ‘PAM’. PAM sequences occur so frequently (about every 50 bases) that we are able to target almost any specific gene. Cas9 will break H-bonds between the nucleotides, unwinding the DNA double helix. If the DNA at that location perfectly matches a sequence of about 20 nucleotides within the guide RNA, DNA and matching RNA will bind through complementary base pairing. This DNA-RNA base pairing triggers CRISPR-Cas9 to activates its nuclease activity, and it cleaves both DNA strands at the site upstream to PAM. After these DNA strands are cut, a natural repair process will begin as the cell tries to mend the broken DNA. These breaks in the DNA can be repaired by a nonhomologous end joining (NHEJ) or homology directed repair (HDR). NHEJ is faster, and error prone, which hopefully will cause the inactivation of the gene. If it is repaired to the same state as before, though, Cas-9 will cleave the DNA again. This process will go on until the DNA is repaired with a mutation that inactivates the target gene sequence.
   The HDR repair mechanism is less error prone, and uses a homologous DNA template to accurately repair the break. Scientists will manipulate this repair system by introducing an excess of the DNA repair template (donor DNA), which tricks the cells repair machinery. The repair machinery will instead use the correct DNA repair template, fixing the harmful mutation in the gene. CRISPR-Cas9 holds a lot of promise, launching us into a new era of Biotechnology. CRISPR-Cas9 can be used to our benefit for not only helping fight harmful mutations that happen within the DNA of humans, but other animals and plants. This possibly mean the reduction of deadly genetic deficiencies and diseases in the near future.

   3. The models we used for this activity helped our learning process along by providing us a visual image of what CRISPR-Cas9 does. These models are handy, as they accurately reflect quite a few of the processes. For starters, the paper model helped show the general positioning and shape of the CRISPR-Cas9 protein. It fell short, however, in multiple ways. The size is (obviously) not accurate, and the use of paper made the model 2D, lacking 3D elements like the fact the DNA is coiled. We also don’t really get to see the specifics of how the DNA is repaired or cleaved, because we are just inserting and cutting out pieces of paper. The paper model does a good job of describing a very general concept, but not the specifics. A change that could be made to this model would be to make it 3D. This would help make the shape a lot more accurate. The digital model we used was very helpful and cleared up a lot of the discrepancies from the paper model. It was very specific with the process descriptions, and 3D. It was also animated, showing the direct way different processes happen. This model can misrepresent the process in a couple of ways; for example, the color coded structures which are not realistic but merely there for the benefit of a learner. Some ways this model could be made better are by allowing free rotation to view different parts of the process from different angles.

   Using models to communicate scientific concepts to non-scientific audiences is incredibly effective. Every model will come with its slight misrepresentations, that’s just a fact. As long as these misrepresentations are minimal in comparison to the bigger topic being discussed, I don’t see the problem. After all, models help us understand the general idea of what we are learning; specifics can be left to the people who want to take that deeper dive. Models especially appeal to visual learners, like myself, because they take conceptual ideas and put them into images we can process. For educating non-scientific audiences, models work just fine.

   Primary Source: CRISPR-Cas9 (hhmi.org)
   Secondary Source: https://www.youtube.com/watch?v=2pp17E4E-O8