Category Archives: Portfolio

One technique that I learned more about in BIOL 463 is CRISPR/Cas9, which stands for clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9. Based on a mechanism of adaptive immunity in prokaryotes against viruses, CRISPR/Cas9 is used by researchers to edit genomes in cells. Essentially, the CRISPR/Cas9 system aims to induce site-specific double-stranded breaks in the genome of cells to allow homology directed repair after adding a DNA template consisting of the modified sequence which we wish to incorporate.

If someone were to learn how CRISPR/Cas9 works, I think the most difficult aspect of this technique to understand is that the single guide RNA (sgRNA) used to target the DNA sequence of interest must consist of both CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). Although the crRNA component of the sgRNA is what actually binds to target DNA by complimentary base pairing, the crRNA component is not sufficient to allow cleavage to occur by Cas9. As CRISPR/Cas9 is famous for its ability to allow site-specific modifications, students who first learn about CRISPR/Cas9 may become too focussed on the aspects that allow specific targeting, such as crRNA, and may overlook another aspect that is not involved in targeting DNA, but is necessary for the CRISPR/Cas9 system to work, such as tracrRNA, especially when the two components are part of a single RNA construct.

If I were to test a person’s understanding of how CRISPR/Cas9 works, I would ask: Why is a DNA template necessary to allow genome editing to occur through CRISPR/Cas9? In an answer, I would expect the person to acknowledge that without a DNA template, double-stranded breaks in the DNA will lead to non-homologous end joining, which results in the incorporation of random bases, and thus random mutations. Purposeful genome editing involves specific modification of sequences, rather than random mutagenesis. Therefore, a DNA template is necessary to allow specific modification of target DNA sequences.

As I was studying for midterm #1, one thing that that stood out for me was that the practice midterm was focused on problem solving, rather than memorization. Before I attempted the practice midterm, I reviewed all of the lecture slides in order to remind myself of the content that had been covered and to re-answer questions that were on the slides. At this point, my aim was to memorize what we had learned in lectures and to practice data interpretation and problem solving.

Then, I attempted the practice midterm. When I looked through the questions, there were fewer questions about the content presented in lectures than I expected. I had anticipated that there would be questions about the honeybee papers, but instead, there were questions about an imaginary organism. Thus, I realized that the questions that we were given for practice were meant to help us practice applying concepts and solving problems, rather than memorizing facts presented in class.

After completing the practice midterm, I decided to adopt a different study strategy. Instead of trying to memorize facts, I focused more on answering practice questions. By doing this, I hoped that my studying would be more effective. However, after completing the actual midterm, I felt that I put less emphasis on memorization than I should have. Although the actual midterm was focused on problem solving and interpreting data that we had never seen before, some of the problem solving required knowledge of techniques. I think I made the right decision to focus less on memorizing facts in general, but I should have put more emphasis on memorizing techniques in order to more effectively answer questions that ask to propose an experiment. By shifting my study strategy, I wanted to optimize my studying by focusing on what I thought was most important (answering questions), but I put a too much attention into one aspect of studying, which led me to have gaps in other aspects. For the next midterm, I will aim to more carefully evaluate what the most important concepts are in order to make my studying more balanced which will hopefully allow me to perform better.

One concept that I understand in a new way is data interpretation. In particular, I am starting to be more thoughtful about what can and cannot be concluded from data.

I began to think differently about the concept of data interpretation when we studied the honeybee papers in class. In one paper, we observed data that showed that siRNA for DMNT3 in L1 honeybee larvae was sufficient to promote the development of mostly queens.  From this observation, I initially inferred that this meant that decreased DNA methylation in L1 larvae would lead to queen development. However, this data alone did not directly show what I had assumed, which made me realize that I could not make conclusions based on assumptions, even if the assumptions seemed logical. In order to validly conclude that decreased DNA methylation is correlated with queen development, we would need to directly measure DNA methylation levels in larvae. Through this experience, I learned that conclusions require direct supporting evidence without having to make assumptions in order to be rigorous.

I think that having a good understanding of the concept of data interpretation is important to ensure that conclusions are valid. Although it is tempting to make extended inferences based on logical assumptions, we need to keep in mind that we can only make conclusions based on the evidence that we have. If assumptions are used to make conclusions, these conclusions may be inaccurate.  Therefore, by making precise conclusions that are based only on direct evidence, we can be certain that our conclusions are reasonable.

Once piece of factual knowledge that I acquired since the start of the course is that the “add something” experimental approach is a test of sufficiency for a result that we wish to induce. For example, we may want to know if adding certain transcription factors to differentiated cells can induce the cells to become pluripotent.

This new piece of knowledge fits into what I already knew by providing an additional method of studying the functions of genes. Previously, I was more familiar with the “remove something” approach for studying genetics because I volunteer at a lab where experiments are often done by using a wild-type strain as a control group and a knockout strain as an experimental group. In my experience, I have never used the “add something” approach in the lab, so I consider it to be an interesting alternative way to perform experiments.

One thing that comes to mind when thinking about the “add something” approach is using CRISPR as a means to do the “adding”. I often hear about how CRISPR can be used to add or edit genes, but I do not know very much about how CRISPR actually works. I would be interested in learning more about CRISPR in BIOL 463.

In my opinion, the “add something” approach fits into the general concept of using experimental approaches to obtain information. Earlier in this journal, I mentioned the “remove something” approach as another experimental approach used to obtain information. Furthermore, in class, we also discussed the “look” approach as yet another experimental approach. Thinking about the concept of experimental approaches in general, I am curious about other approaches that may be possible which allow scientists to obtain information in different ways. Just like how the “add something” approach is a test of sufficiency and the “remove something” approach is a test of necessity, I wonder how other approaches can provide information in other ways.