CRISPR modelling Edublog post

CRISPR-Cas9 (often shortened to “CRISPR”) is a biotechnology tool that can be used to edit the DNA in cells and organisms. By using our paper model of the system, we were able to investigate how CRISPR-Cas9 edits DNA. To understand how CRISPR-Cas9 is utilized to inactivate (“knock out”) genes and to more precisely alter their sequence, we employed the paper model. Next, we looked at a CRISPR-Cas9 online model and saw how this technology is currently being utilized in research.

CRISPR-Cas9 is an acronym for “Clustered Regularly Interspaced Short Palindromic Repeats” and “Cas9” for the CRISPR-associated protein 9. The system of CRISPR-Cas9 is made up of two fundamental components; the cas9 protein and the guide RNA (gRNA). The guide RNA is there to help CRISPR identify which gene it’s specifically looking for and once it finds that gene the Cas-9 cuts the gene out. These two parts work together to help achieve the overall goal of editing out the unwanted gene. When faced with a specific gene target the CRISPR Cas-9 when released, will go along a strand of DNA and scan the strand for the PAM that’s associated with the gene they need to target.  When the correct PAM is found the CRISPR molecule binds with the PAM.

In the model above, the blue section of the guide RNA represents the part that binds to the Cas9 enzyme. The red section of the RNA, which has a sequence written out, represents a “targeting sequence” that is free to bind to a complementary DNA sequence.

CRISPR-Cas9 binds to the target area of the specific gene by recognizing the three-nucleotide sequence called PAM, which occurs throughout the genome. Once Cas-9 binds to a PAM sequence it unwinds the DNA if the guide Rna matches the DNA sequence next to the PAM, the guide RNA will bind to the complementary DNA strand. If the guide RNA doesn’t match the DNA sequence CRISPR leaves the DNA alone and allows it to zip back up.

Once the guide RNA binds to the DNA, it activates nuclease activity also known as DNA’s cutting ability or “cleaving”. CRISPR-Cas9 cleaves DNA by having the target DNA and the guide RNA form a helix shape once they bind together. This binding activates the DNA’s cutting activities. The Cas-9 created cuts three nucleotides above the PAM and cleaves the DNA strands. This results in a double-stranded DNA break.  Shown below↓

CRISPR-Cas9 can repair DNA to “knock out” a gene by introducing small insertions or deletions through non-homologous end joining (NHEJ), which disrupts the gene’s coding sequence. Two main repair mechanisms may occur: one is a frameshift mutation, which alters the gene’s reading frame, resulting in nonfunctional proteins; the other is premature stop codon creation, which halts protein synthesis early, inactivating the gene. This leads to a “knockout” effect, where the gene no longer produces a functional protein, effectively silencing its associated trait. For example, CRISPR-Cas9 can be used to generate knock-out cancer cell lines. In these cases, a single guide RNA (gRNA) often induces an insertion or deletion to create knock-out cells. However, some cells may still express the target gene by skipping the disrupted exon or using an alternative splicing variant, thereby bypassing the targeted exon.

CRISPR-Cas9 can repair a mutation in DNA by working to find a targeted DNA sequence and knocking it out to protect it from mutations or infections. When it finds the target DNA it binds to the strand and cuts it open to have better access to the affected area. From there it cuts out the unwanted part and puts it back together. This process of cutting out specific nucleotides is useful when someone has a mutation in their DNA.

CRISPR-Cas9 could be used to our benefit by using this technology to save people, plants, and animals from harmful viruses and infections. An example of this is if crops were affected by an infection that resulted in lots of wasted crops we can use CRISPR Cas-9 technology to target that gene or DNA sequence that’s affected and knock it out of the DNA sequence.

The model effectively represented the CRISPR process, helping us visualize and understand how it works. By using the model, we gained a clearer sense of how different components interact and also got a basic idea of each part’s shape. However, the model can also create some misrepresentations. One issue is with scale—because CRISPR is extremely small, we need larger models to see the parts clearly, which might lead people to think CRISPR itself is larger than it actually is. Another potential misrepresentation is the shape, as the model can give an incomplete view; CRISPR, like many biomolecules, is three-dimensional, yet models often appear two-dimensional. This can create a misconception that CRISPR is flat.

Improving the modeling activities, perhaps by introducing 3D models, could better reflect the actual CRISPR process, particularly benefiting hands-on learners who grasp concepts by seeing and manipulating full diagrams. While models are a valuable tool for helping beginners or younger students understand complex topics, they may not be as helpful long-term. Initially, models allow students to create and visualize the process, but as they advance, relying too heavily on models may lead to misunderstandings about features like size, shape, or color.

SOURCES

• https://www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications
• https://pmc.ncbi.nlm.nih.gov/articles/PMC9850488/
• anatomy and physiology textbook pg #72 – 81
• https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2019.00551/full
• https://www.nature.com/articles/s41467-024-48598-2
• Doudna, J. A., & Charpentier, E. (2014). “The new frontier of genome engineering with CRISPR-Cas9.” Science, 346(6213). DOI: 10.1126/science.1258096.
• Hsu, P. D., Lander, E. S., & Zhang, F. (2014). “Development and Applications of CRISPR-Cas9 for Genome Engineering.” Cell, 157(6), 1262-1278. DOI: 10.1016/j.cell.2014.05.010.
• Cong, L., et al. (2013). “Multiplex Genome Engineering Using CRISPR/Cas Systems.” Science, 339(6121), 819-823. DOI: 10.1126/science.1231143.
• Jinek, M., et al. (2012). “A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity.” Science, 337(6096), 816-821. DOI: 10.1126/science.1225829.