1. An introduction to the topic and how you’ve been exploring it 

-CRISPR-Cas9 (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeat) is a genetic editing tool that allows parts of DNA code to be changed/altered. This is being used in the scientific and health community to help cure diseases and remove unwanted sequences of DNA that are harmful. It does this by genome editing and the Cas9 enzyme which is the enzyme that cuts the DNA and helps edit DNA sequences.  It is used because firstly, it is relatively cheap compared to alternate methods of genome change/editing. It is quick, efficient, and accurate when it comes to editing. It does not cause pain when doing so. Also, it can help cure and or help prevent diseases around the world if used correctly. It could help cure cancer for good if developed in the long term.

-On the image below you can see each individual part. The squidward face glob is the Cas9 enzyme and it is used to alter parts of DNA code.

-The blue strands are both RNA 1 and 2 (NHEJ and HDR). The reddish pink strands are the DNA code (DNA triplets and RNA Codons). The yellow highlight upon said strands represents PAM which will be described further down this Edublog post.

-The small red rectangular paper represents the Donor DNA which is used only during the final step.

-The small green square represents random nucleotides which also is only used during the final step.

2. Explain the entire process of DNA editing using CRISPR-Cas9, including:

The three steps of CRISPR -Cas9 DNA editing are: recognition, cleavage, and repair.

CRISPR stands for, Clustered Regularly Interspaced Short Palindromic Repeat, which are the steps and structure describing the CRISPR editing.

It is used in DNA editing because the Cas9 enzyme, which is produced by CRISPR, is able to bind to targetted parts of DNA and cuts it off changing the sequence and altering the entire thing. This is able to do quick and easy changes that can save lives and help cure diseases.

Below is the Cas9 Enzyme. That is what its structure looks like. (ignore the RNA in it I didn’t have a better photo of just the Cas9 enzyme).

The first step to CRISPR editing is RECOGNITION. 

This is the step where the Cas9 RNA recognizes and binds to the targetted part of DNA that it will be cutting. CRISPR-Cas9 creates RNA with a guide sequence that binds to the specific target DNA sequence. The first part if the RNA binding to the Cas9 enzyme. Then following the DNA binds to Cas9 enzyme right below the RNA. This leads to the RNA binding to the targetted sequence of DNA which will later be removed from the sequence resulting in a mutation.

RNA binds to enzyme below:

In the photos below you can see the RNA binding to the targetted DNA sequence.

In this step we can see the PAM (protospacer-adjacent motif) which marks the targetted spot (highlighted in yellow above). The RNA binds a top of the DNA as shown in the model below. In the CLEAVAGE step we can see another reason why PAM is so important when it comes to CRISPR editing.

By the end of this step the RNA and DNA have both binded to the Cas9 enzyme and the RNA has successfully binded to the targetted part of the DNA sequence.

The second step to CRISPR editing is CLEAVAGE. 

This step describes the cutting of the targetted sequence that results in change/alterations. After both the RNA and DNA have binded to the Cas9 enzyme this step can proceed. After the first step the RNA is “programmed” to find the targetted DNA because of the guide sequence.

In the photo below you can see the Cas9 enzyme using a specific DNA sequence called PAM (protospacer-adjacent motif) which is used to mark the targetted DNA site. The use of PAM allows the CRISPR to distinguish the target from other parts of the sequence so it does not cut the wrong bit. It binds to the guide RNA at the top (blue strand) and then proceeds to cut/cleaves both strands of DNA. So not only does PAM mark the target sequence, but it also allows for the CRISPR-Cas9 to distinguish between the target and other parts so it does not miss cut the wrong part. If it did cut the wrong part the entire DNA mutation alteration would be different from the one intended. Though our group forgot to properly model the cleavage step, here is the image from our instructions that demonstrates it. The scissors are cleaving/cutting it. 

After this step the CRISPR-Cas9 can insert or delete strands entirely which leads to a complete editing of DNA and the creation of the wanted mutation.

The third step to CRISPR editing is REPAIR. 

The REPAIR step is when the DNA sequence that was cut is repaired and fixed by either removing the targeted DNA sequence and then reconnecting the seperated parts and or inserting a new DNA sequence where the targetted one was removed. These two different changes are called Gene KNOCK-IN or KNOCK-OUT. After the Cleavage of the targeted DNA sequence cellular enzymes will attempt to repair this.

We modeled both applications of CRISPR-Cas9 repair mechanisms.

The first one below is Gene Knock-out which is used to inactivate a target gene (Nonhomologous end joining or NHEJ). Unfortunately, this method is more prone to errors because it does not use a template unlike the next editing mechanism. there This where a random nucleotide is used which represents the mutation that will inactivate the targetted gene/sequence. The green paper represents the random nucleotides. It is called a gene Knock-out because when the random nucleotides are added they repair by knocking out any other sequences that will activate it. This therefore inactivates it.

The second model below represents Gene KNOCK-IN where a “donor DNA” is used as a template to trick the cell into using HDR (homology directed repair). This mechanism is much less prone to error making it a safer choice. This is used to replace a mutation with a new sequence thus the name “Gene Knock-In”.

The two images below represent the repairing of DNA.

The image below represents the use of a Donor DNA as a template. This leads to the change in sequence being less prone to error because it uses a template to go after.

The gene KNOCK-IN and KNOCK-OUT are very important to changing mutations because they can successfully alter an entire sequence of code by either knocking-in a new gene (insertion) which changes the whole sequence (frame-shift mutation) or knocking-out a gene by using a DNA template for the code to copy therefore changing the sequence. These can help knock-out a mutation like say heart disease which kill around a million people in the world yearly.

CRISPR-Cas9 can be used for our benefit because:

Using CRISPR’s three steps, RECONITION, CLEAVAGE, and REPAIR, people are able to change sequences within their cells which can therefore help save lives. That is how CRISPR-Cas9 can repair and change mutations that could potentially harm us. It can either insert or delete the targetted mutation which changes it for the better. With the use of CRISPR people can afford this quick and easy change in their DNA. This technology can save lives by changing sequences of DNA code. It can be used to cure cancer and other very harmful diseases. It can be used as an alternative that would not cost as much money as other treatment plans. Not only that but, it is a very fast process which may lead to faster and successful developments of treatments for fatal or harmful diseases and viruses. 

3. In the CRISPR modelling activity, we used models to learn about a complex microscopic process (paper cut-outs, digital simulation).

-The models we used accurately reflected the process relatively well because it was simplistic in a way that a grade 12 Biology student could understand, but not too simple that it did not appropriately describe the process/steps. Though it would be a bit better if we had a model that we could see physically changing in front of us rather than changing the paper around to demonstrate this change. For instance, a model replica online probably would have shown us the steps significantly better than this, but using this method we could change it ourselves which helps show our learning. In conclusion, I think it was a great way of showing and demonstrating our learning of CRISPR-Cas9 editing and why it is so important for the scientific world.

4. Models are commonly used to communicate scientific concepts to non-scientific audiences. Do you think this is an effective way to educate students and/or the public about science? Explain why or why not.

-I believe it depends on the model because some help show how the biological thing would look while other just confuse the person more. For instance, organelles within a cell drawings are helpful models because they show what they look like, but they do not communicate the fact that they are not completely stationary at all times because the paper is static. This would be an example of a great model that shows information in a clear and concise way. Though the image is a bit blurry because it is not very big, it shows each individual organelle of a plant cell. You can clearly see each part and no arrows are crossing over one another. The different parts are colour coded so you can tell them apart very easily and even someone who has never taken a biology class can grasp at the different parts, what they look like, and how they differ from one another.

Unfortunately, for bad models they fail to represent the information they have drawn out. Like this model I found on the internet. My issue with this model is that it communicates way too much information in a non-neat way. There are so many arrows and they distract you from the main parts. Someone who did not take biology would see this and freak out because there are so many different parts. Why show the inside of an animal cell if you are also then going to show so many different dendrites everywhere on the page. It draws your eyes from one place to another and you do not even consume the information. Some of the writing is even covered by the drawing. A model must demonstrate information in a neat and tidy way to be great.

The use of models to educate the public on science can be great as long as it is done in such a way that you can view the information well, it is legible, and tidy. If able to a legend describing specific zones would be very useful for those struggling to grasp the topic. Arrows should not crossing over one another. To sum up, models are good ways to demonstrate knowledge even if done on a static piece of paper. They can educate and teach people biological things (muscles, cells, bone structure, etc.) without needing to look inside of an actual body or microscopic example. They can be done in a tidy and neat way as to show people without stressing them out.

Models should be continued to be used throughout the teaching world to educate individuals about science!