Catherine's Blog

The CRISPR-Cas9 is peaking interest in the world of biotechnology. It is a process being explored and used to modify genetic information. CRISPR-Cas9 is a step into the future of biotechnology and all sorts of genes.  But how does it work?  We have built a model and explored simulations to understand this revolutionary process. It is truly something fascinating and worth researching more into.

What does CRISPR-Cas9 mean?

CRISPR stands for: Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR is a segment of DNA that contains short repetitions of base sequences which is involved in defense mechanisms in prokaryotes towards viruses. Cas9 stands for: CRISPR-associated protein 9. It is an enzyme that cuts nucleic acids and works with CRISPR in order to create revolutionary gene-editing technology. The CRISPR-Cas9 simply makes it possible for the corrections of errors within the genome by turning genes on or off with relative high speed, low cost, and ease.

Cas9

CRISPR-Cas9

Structure of CRISPR-Cas9:

The CRISPR-Cas9 system is made up of two components: Cas9 protein and guide RNA. The Cas9 is an endonuclease enzyme and the guide RNA is a synthesized RNA molecule. In the picture above, we can see how they fit together.

How CRISPR-Cas9 targets a specific gene:

Scientists design a specific Guide RNA (gRNA) to introduce to the Cas9. A common analogy used is to think of the Cas9 as scissors and the gRNA as a programmable GPS. The gRNA is synthesized to contain a sequence of 20 amino acids to match a specific sequence in the cell's DNA to target it. Once the gRNA is bound to the Cas9, it guides the Cas9 towards its' target. The target sequence can be any sequence as long as it is near a Protospacer Adjacent Motif (PAM). PAM is usually a three-nucleotide sequence that consists of 5'-NGG-3', where 'N' represents any nucleotide base (A, C, G, T) and 'G' representing guanine. Since PAM motifs occur about every 50 bases or less in humans, this allows us to use the Cas9-guide RNA complex to target almost any gene.

How CRISPR-Cas9 binds to the target area of the specific gene:

The Cas9 recognizes and binds to the PAM motif in the cell's DNA. After binding, the Cas9 unwinds and unzips the DNA's double helix. If the DNA sequence is not exactly complimentary to the gRNA, the Cas9 disengages and moves on as the DNA rezips and rewinds. If the sequences match, the complimentary base pairs form a DNA-RNA helix.

How CRISPR-Cas9 cleaves DNA:

After binding, the Cas9's nuclease is activated. It makes specific cuts in the DNA, three nucleotides up towards the 5' end from the PAM site. There are two active cleavage sites on the nuclease to generate the cuts to cleave both strands of the DNA's double helix. As a result, there is a double-stranded DNA break.

How CRISPR-Cas9 can repair DNA to “knock out” a gene:

After cleaving, cellular enzymes attempt to repair the break, allowing CRISPR-Cas9 to take advantage of their mechanisms and alter the target gene. More often the cell uses Non-Homologous End-Joining (NHEJ) to repair the cell as it is faster since it does not use a template to join the broken DNA ends together. However, this mechanism is error-prone as it may introduce mutations within the target sequence. Errors are rare, so when the break is correctly repaired, the Cas9 recognizes the target sequence again and cleaves it. The repeated cycle eventually causes a random mutation within the desired sequence. If the target is within the gene's coding region, the mutation will inactivate or 'knockout' the gene.

How CRISPR-Cas9 can repair a mutation in DNA:

On the other hand, if the cell uses the Homology-Directed Repair (HDR) approach, the CRISPR-Cas9 is able to edit the target gene. This mechanism is less error-prone since it uses a template to accurately repair the break. Scientists are able to manipulate this mechanism by inserting excess of a DNA repair template into the cell alongside the Cas9-gRNA complex. This causes for the cell to be tricked and to use the template to complete HDR. With varying repair templates, scientists are able to change the target DNA sequence into a new sequence. These templates can correct existing mutations by replacing it with a nonmutated sequence of DNA.

How can CRISPR-Cas9 be used to our benefit?

Biotechnology and gene-editing have been around for a long time. However, CRISPR-Cas9 is believed to be revolutionary as it is an efficient, cheap, and fast way to alter genes. It is said that CRISPR carries the potential to change anything the involves genes. Its' potential is vast, from agriculture to curing diseases. Numerous scientists believe that it may lead to great breakthroughs in fighting against cancer, HIV, and heart disease. There are many ongoing studies and trials in order create a reality out of the potential. There has already been successes as CRISPR has been used to treat a genetic heart defect in human embryos. However, this method is relatively new and scientists are still discovering how interconnected DNA really is and the possible effects on other genes.

How the model accurately reflected the process:

The model was able to accurately demonstrate the big ideas and key parts of the process. We are able to see what the result of each step looks like and grasp a fundamental understanding of the CRISPR-Cas9 process.

How the model misrepresented the process.:

With a 2D model, there is only so much we can visualize. For starters, we are unable to experience the actual build of the components which takes away some of the real complexities within the system. Furthermore, we miss the transitions between the key stages hence, we have a decreased understanding of how the components act.

How to improve the modelling process:

I found it much simpler to visualize the system once I used the digital simulation. I feel that it did a good job at filling in the missing pieces in transition and throughout the stages. For a class activity it is less hands on however, it is much more accurate. Overall, I feel that we should use the tools that we have to their full extent.

Are models an effective way of educating?

There are many different ways that people are able to learn and understand material. I feel that models are great when it comes to visual and physical learners. When using models, I feel that they are much more stimulating as we are pushed to learn actively and not fall into a passive state. The more senses we expose our learning to, the deeper the understanding we are able to grasp of the topic. Furthermore, if certain senses are weaker at retaining information, there are other senses that are able to back it up. Overall, I find that models are one of the most effective educational techniques.

Sources:

“Building a Paper Model of CRISPR-Cas9.” HHMI BioInteractive, 13 Apr. 2020, www.biointeractive.org/classroom-resources/building-paper-model-crispr-cas9.

CRISPR-Cas9, media.hhmi.org/biointeractive/click/CRISPR/?_gl=1%2Aoyzf8c%2A_ga%2AMTY3OTA0NTYyOC4xNjk1NzY1MDUw%2A_ga_H0E1KHGJBH%2AMTY5NzIxMDQ1NC4xMC4wLjE2OTcyMTA0NTQuMC4wLjA. Accessed 21 Oct. 2023.

“The Now: How CRISPR Could Change the World.” GCFGlobal.Org, GCFGlobal Learning, edu.gcfglobal.org/en/thenow/how-crispr-could-change-the-world/1/. Accessed 21 Oct. 2023.

Redman, Melody, et al. “What Is CRISPR/Cas9?” Archives of Disease in Childhood. Education and Practice Edition, U.S. National Library of Medicine, Aug. 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC4975809/#:~:text=Clustered%20regularly%20interspaced%20palindromic%20repeats,cheaply%20and%20with%20relative%20ease.

webteam), www-core (Sanger. “What Is CRISPR-Cas9?” @yourgenome · Science Website, 8 Feb. 2022, www.yourgenome.org/facts/what-is-crispr-cas9/#:~:text=The%20CRISPR%2DCas9%20system%20consists,then%20be%20added%20or%20removed.