In Anatomy and Physiology 12, we’ve explored the fundamental functions of nucleic acids and how genes work. DNA, an essential part of our cells as it withholds vital genetic code to produce proteins, is also where genetic mutations occur because of faults within the replicants of its code. These errors, which occur during DNA replication, cause changes to the sequence of the amino acids that form polypeptide chains; altering the protein’s function. As we’ve learned in the last Biomolecules unit, proteins and their functions are essential to the body; like Collagen, which provides structural support in various tissues, and the proteins Actin and Myosin, which enable movement within our muscle cells. With genetic mutations, it all depends on how the function of the mutated protein plays out to be; it can be very harmful and severely impact a living thing’s quality of life, or it can be beneficial and improve it significantly, or, well, it may just neutral, which is what most genetic mutations are. With evidence of harmful genetic mutations, such as the scientific community is constantly working on something called DNA technology, which has come to develop vital biotechnological tools such as the CRISPR-Cas9 editor.

The CRISPR-Cas9 consists of two main components; CRISPR and Cas 9. CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. To put all those confusing words into sense, this component is essentially a segment of DNA containing short, repetitive sequences- the same sequences of a bacteria’s DNA genome would have. CRISPR serves as a memory of past viral infections and allows the bacterial immune system to recognize and defend against them. This system was first discovered in the immune system of bacteria and is how bacteria recognize and cut DNA sequences to prevent viral infections. In part of the CRISPR-Cas9 biotechnological tool, scientists have used this system and Cas9, an enzyme, to cut out DNA in specific locations.

 

 

 

 

 

 

 

The 5 steps of the CRISPR-Cas9 mechanism:

1. As a ‘gene knockout’ that will inactivate or disable the target gene using non-homologous end joining, or NHEJ. This is a bit of a faulty repair mechanism. In this activity, this repair mechanism is modelled with the random nucleotide, shown in green, taped between the two pieces of the cut target DNA.
2. The second mechanism is homology-directed repair (HDR) which is used for precise edits to the target gene with a provided “donor DNA” that serves as a template that will trick the cell into ‘knocking the gene in’. This mechanism is more robust than the NHEJ and can be used to replace a mutation with a normal type sequence or to just add a new one altogether. In this activity, we modelled the mechanism by removing the 1st gRNA and target DNA from the Cas9 enzyme then repeating the targeting, binding, and cleaving using a secondary RNA and target DNA. Through using the real mutant version of a gene called MYBPC3, which causes a deletion that results in a type of heart disease, we modelled the outcome of HDR by placing the “Donor DNA” piece on top of the cut pieces of the secondary target DNA. We then taped the Donor DNA over the target DNA pieces (not pictured).

Analyze the models you used to learn:

The 2D model accurately portrays the lining up of the gRNA as it targets and binds to the gene template- the visual clues of the yellow PAM sequence really helped, as well as the cleaving that was outlined where it needed to be for us at the 5′ end by a dotted line, done by the Cas9 (this was especially useful in the way it as modelled in the activity because we physically cut the strands). I would say that the biggest misrepresentation of the paper model was that it was unclear as to where everything was located within the cell in the 3D sense, as well as how it was in portion to other things (the Cas9 is, of course, not that up to scale). All the while, the online Interactive Exploration enabled a better overall explanation of the entire procedure of CRISPR-Cas9 and its mechanisms, such as in the greater detail given about the unwinding of the DNA strand.  While I do love the hands-on and tactile aspect of the 2D model, I would have wanted details about each label of the different parts of the model; for example, if on the back of the gRNA strand there was a definition of the sequence rather than on a separate piece of paper (like in the activity). It would have also been beneficial to have seen a variety of different genes onto which we could use the CRISPR-Cas9 tool, such as a couple within each of the two repair mechanisms. Overall, I would say the combination of both the Interactive Online Simulator and the 2D paper model was an effective way to get the idea across. Hopefully, with more easily found information on the back of the paper cutouts, it would be even easier and more accessible to non-scientific audiences.

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