In the past decade biotechnology has been making incredible scientific breakthroughs, including the development of the genetic engineering technology CRISPR-Cas9. So, what is CRISPR-Cas 9 and how does it have the potential to revolutionize a number of scientific fields? That is what I will be answering below as I describe the entire CRISPR-Cas9 DNA editing process as part of my own investigation.

So, what does CRISPR-Cas9 mean? CRISPR stands as an acronym for Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 stands for CRISPR-associated protein 9, which is an endonuclease, meaning it is an enzyme that cuts DNA. CRISPR-Cas9 is an incredible advancement in genetic engineering and allows for precise DNA modifications in living things. The structure itself consist of two main components: Cas9 protein and guide RNA which are the basic building blocks. Given its ability to cut DNA at very distinct locations, Cas9 is frequently compared to a pair of molecular scissors. Guide RNA, on the other hand, functions like a molecular GPS system. It guides Cas9 to the precise molecular address or the particular region in the genome that scientists want to change. This interaction between Cas9 and guide RNA allows for exact targeting and editing of specific genes making it such a crucial tool in the field of genetics

So how does CRISPR-Cas9 target a specific gene and bind to it? Well as said before CRISPR is able to target specific genes through its interactions with guide RNA. The guide RNA is synthesized by scientists consisting of about 20 nucleotides that match a particular sequence in cell DNA of which they want to target. When introduced into a cell the guide RNA actively seeks out and attaches to the precise region within the target gene, helped by the presence of PAM. PAM stands for Protospacer Adjacent Motif, and it is a certain type of nucleotide sequence that is usually 5’-NGG-3’ where N represents any nucleotide (A, C, T, G). PAM sequences occur every 50 bases or less which is why scientists can target nearly every human gene. Once guide RNA successfully guides Cas9 to the target gene, it will bind there. After binding, the Cas9 will “unzip” the DNA double helix, if the unzipped DNA strand is not an exact match to the guide RNA the Cas9 will disengage and zip back up the DNA. If the DNA is a perfect match the guide RNA will bind with the single DNA strand and form a DNA-RNA helix

After binding, Cas9’s nuclease with be activated and start making specific cuts in the DNA, it usually begins 3 nucleotides upstream from the PAM site, this is called cleaving. On the nuclease part of Cas9 are two active sites that cut and cleave both strands of the DNA double helix, making it possible for a complete double-stranded DNA break

When DNA is broken it can be repaired by either a nonhomologous end joining (NHEJ) or by a homology-directed repair (HDR). NHEJ is more frequently used as it’s a fast repair mechanism and tends to be error prone. NHEJ happens when specific cell enzymes repair the double stranded DNA with nucleotides, Cas9 will then recognize the DNA sequence and cleave it again. This cycle continues to happen multiple times and will eventually result in a mutation. The mutation will be random but if it occurs exactly at the targeted sequence it will likely inactivate that entire gene. HDR is when a DNA template is introduced in order to “trick” the cell into using the synthetic template (aka Donor DNA) to fix the DNA break. When designing the repair templates scientists can change the target DNA sequence into a whole new sequence. These synthetic templates are also used to correct pre-existing mutations by replacing it with a non-mutated DNA sequence. These two methods of DNA repair are also called gene “knock-out” and provide scientists with the ability to further understand gene functions and create potential therapeutic interventions, whether in research or medical applications

Overall, CRISPR-Cas9 has shown great potential in reversing genetic mutations. It has the potential to revolutionize both scientific research and practical use with its unique ability to target specific genes. CRISPR-Cas9 potentially offers a way to tackle inherited diseases at the genetic level. It has the ability to target and treat the genetic mutations that underlie inherited diseases like cystic fibrosis. It can provide treatments for cancer by addressing particular genetic mutations. Or it can improve resistance to diseases and also lower health risks associated with genetic factors like heart disease. CRISPR-Cas9 has plenty of possible uses, and furthermore is not just used for medical purposes; it is also changing the agricultural field by making it possible to generate crops with improved features.

For this activity we used both physical paper cut outs and an online simulation to investigate and gain understanding of the complex CRISPR-Cas9 microscopic process. Each model had both their advantages and disadvantages. To start off the paper model was easy to follow along with and correctly portrayed the crucial roles of target DNA, PAM, guide RNA, Cas9 protein, donor DNA, and nucleotides in the CRISPR-Cas9 process. However, compared to the online simulation the paper cut outs were simplified and left out many details of the process. The online simulator also was able to represent the dynamic three-dimensional interactions that takes place during the process, which is a part the paper cut out activity lacked in. With the online simulator, a person had the advantage of “exploring” the website in an interactive way. It provided simple definitions of key terms and also went into depth when explaining the four main steps in a CRISPR-Cas9 process. A potential disadvantage is that the online simulation takes longer to go through and thoroughly understand than the paper cut out activity does. A potential change for both models we used could be for them to provide more visual detail when it comes to how the Cas9 specifically cleaves the DNA strands, as I found that both sources had very limited visuals and information for that crucial step.

I think models are an effective way to educate students about science. Models are a helpful tool for explaining scientific ideas to people who are not scientists and do not have an in-depth knowledge of certain scientific topics. They provide a clear and approachable way of explaining difficult concepts. While they may not substitute for comprehensive scientific expertise, they serve as an excellent starting point for students and the general public to grasp the basics of a scientific concept. Models make complex processes simpler and encourage participation, which makes them an important educational tool for teachers to use in their classrooms in order to try to close the understanding gap between complex scientific concepts and general knowledge.

Sources:

Asmamaw, Misganaw, and Belay Zawdie, “Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing.” Biologics : Targets & Therapy, U.S. National Library of Medicine, 21 Aug. 2021, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388126/#:~:text=The%20mechanism%20of%20CRISPR%2FCas,through%20a%20complementary%20base%20pair.

“CRISPR-Cas9 Mechanism & Applications.” BioInteractive, hhmi, 11 Apr. 2018, https://www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications?classId=720356c3-6af0-46b1-8905-736e29b02c2d&assignmentId=c29a5fb0-b3a0-4a8a-8d5a-6247b98c76b2&submissionId=b5da0e2c-4ed0-739b-6ad6-55bda9c67ab4

Liu, Wenyi, “Applications and Challenges of CRISPR-Cas Gene-Editing to Disease Treatment in Clinics.” Precision Clinical Medicine, U.S. National Library of Medicine, 10 July 2021, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8444435/#:~:text=Clustered%20regularly%20interspaced%20short%20palindromic,biotechnology%2C%20and%20human%20disease%20treatment.