Introduction

In our world, there’s a group of microorganisms called bacteria. Bacteria can be quite harmful to human bodies, and they can cause all sorts of sicknesses and symptoms. To fight bacteria, scientists have developed what’s called antibiotics. These work by killing the bacteria or taking away their ability to multiply. However, these little microorganisms are resilient. Antibiotics that may have worked on certain bacteria will not work after a while due to mutation. Bacteria possesses the unique ability to mutate (change) a part of their gene that’s used to be targeted by the antibiotics, effectively becoming immune. This targeted mutation is done by an enzyme called CRISPR – CAS9, or CRISPR for short.

In this post, I will be exploring how bacteria edit their genes using CRISPR based on the information we gained through a step-by-step paper model simulation.

                                  

Figure 1                                         Figure 2                                          Figure 3

So, what is CRISPR – Cas9?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), is a repetitive gene in the DNA. Cas9 is the name of the enzyme (protein) that is closely associated.

The structure?

CRISPR – Cas9 consists of the enzyme, Cas9, and a guide RNA attached to the enzyme as shown in figure one. The guide RNA is coloured blue.

How does it target specific genes?

Cas9 first takes a hold of a guide RNA. This guide RNA then programs the Cas9 to seek out a DNA strand with the corresponding nucleotide sequence. Without the corresponding DNA sequence, the Cas9 will not cause any effects.

How does it bind?

Once Cas9 is programmed to seek out the corresponding DNA, it will begin recognizing and binding to PAM sequences (a three-nucleotide sequence that is repetitive throughout the genome). It then unzips the DNA, and the Guide RNA attempts to bond to the corresponding strand. If the DNA and RNA do not match, the cas9 rezips the DNA and lets go, retrying for a different strand.

How does it cleave (cut)?
Once Cas9 finds a complementary DNA strand to its guide RNA, it cuts through both strands of the DNA 3 nucleotides upstream (towards the 5’ end) of the PAM sequence.

How can it “knock” out a gene?
Once Cas9 creates a breakage in the DNA, cellular enzymes will naturally try to repair the DNA strand. Most commonly, the cas9 will use a method called, non-homologous end-joining (NHEJ). This method allows the Cas9 to join the ends of cut strands of DNA, inserting extra nucleotides and taking out nucleotides along the process as can be seen in figure 3. These extra nucleotides cause a frameshift mutation, potentially drastically changing the product amino acid chain.

How does it “repair” a mutation?
In a similar manner, when the cellular enzymes try to repair the DNA, it can insert or replace a mutation with a normal genetic sequence in a mechanism called homology-direct repair (HDR). In this process, instead of randomly inserting nucleotides, cellular enzymes will copy a template DNA strand into the edited DNA strand.

How can Cas9 be used to our advantage.
By utilizing the CRISPR-Cas9, doctors will be able to cure genetic conditions such as cystic fibrosis by using the NHEJ mechanism. Or, if we find a positive mutation that we might want to incorporate into our DNA, we could use HDR to edit our genetic make-up in an advantageous way.

The modelling processes
The modelling activity was helpful in aiding the visualization of how CRISPR-Cas9 operates. The paper model created an engaging process that allows you to learn step-by-step of each processing step and the purposes behind them. However, since they are on paper, it doesn’t give the participants a complete idea of what the process looks like in 3D (as is in our cells). The digital model did provide a 3D visualization of the “knockout” process, however it only simulated one instance. It might’ve been more helpful if it could show the HDR process as well.

I believe models are a positive asset to learning, especially in science.  Modelling allows people to visualize and study processes more in depth than relying purely on words and taught concepts. Especially now that we have different mediums available (digital, physical, etc.), it not only aids in personal understanding, but also aids in teaching a larger group of people a concept that might be hard to visualize by creating a common ground of understanding.

Extra resources
“CRISPR Knock-in via Homology-directed Repair (HDR).” Integrated DNA Technologies, www.idtdna.com/pages/applications/hdr-for-introducing-mutations.

“Gene Knockout.” Integrated DNA Technologies, www.idtdna.com/pages/applications/gene-knockout#:~:text=How%20CRISPR%20gene%20knockout%20works,Cas%20enzyme%20cuts%20the%20DNA.