CRISPR – Cas9
For the past week in Anatomy and Physiology 12, we’ve been exploring CRISPR-Cas9, a biotechnological tool that allows us to edit an organism’s DNA. In this unit, we’ve learned about mutations—changes in DNA sequences—and how our DNA replicates itself during cell division. Understanding this process is crucial because errors can occur during DNA replication. For example, bases can become mismatched, and if the repair enzyme that checks the DNA fails to recognize this error, it can lead to a mutation. CRISPR is closely related to these mutations because it is a powerful gene-editing tool that can potentially correct errors in the DNA sequence. Since CRISPR is a relatively new concept in the scientific community, we’re focusing on presenting our understanding of this technology. One of our first activities as a group was to create a paper model of the CRISPR system and how it functions. This hands-on project allowed us to visualize the tool’s role in editing DNA, making the concept much easier to understand. To deepen our understanding of CRISPR and its capabilities, we also used an interactive online resource featuring animations explaining each process step. My goal in writing this edublog post is to examine the CRISPR process and its application.
DNA editing using CRISPR-Cas9
CRISPR stands for clustered, regularly interspaced short palindromic repeats, which are repeating sequences found in the DNA genome of bacteria. Cas9 stands for CRISPR-associated protein 9. It is an endonuclease, meaning it’s an enzyme that cuts nucleic acids. CRISPR was first founded to be used as a natural defence mechanism for bacteria when a virus invaded them. When a virus invades a bacterium, the bacterium will capture a small piece of the virus’s DNA and store it so if the same virus invades again bacterium can recognize and target the virus, leading the Cas9 enzyme to cut and destroy the viral DNA.
The process will start with CRISPR targeting specific genes through its interactions with guide RNA. Scientists synthesize the guide RNA and consists of about 20 nucleotides that match a particular sequence in cell DNA that 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.
Cleaving DNA “cutting DNA” begins with the 3 nucleotides upstream from the PAM site. Cas9 unwinds the DNA and uses its nuclease domains to cut both strands, creating a double-strand break.
Cas9 will repair DNA to “knock out a gene”. There are two ways that DNA can be repaired: non-homologous end joining (NHEJ) – more frequently used – or homology-directed repair (HDR). During NHEJ, specific enzymes within the cell quickly repair the double-stranded DNA break by inserting or deleting nucleotides at the site of the cut. When using CRISPR-Cas9, the Cas9 enzyme cleaves the DNA at a targeted sequence, and NHEJ attempts to repair it. However, the repair is often imperfect, and after several rounds of repair and re-cleaving by Cas9, this process can result in a mutation. If the mutation happens at the target site, it may inactivate or “knock out” the gene, preventing it from functioning. Another way that DNA can be repaired is HDR. Through the process of HDR, scientists introduce a DNA template (also called donor DNA) to “trick” the cell into using this template to repair the DNA break. The template is designed to introduce specific changes to the target DNA sequence, allowing scientists to either replace a faulty gene with a correct version or insert entirely new sequences.
Overall, between these two ways that DNA repairs itself, NHEJ is faster, but HDR is more precise.
Benefits of CRISPR – Cas9
This relatively new biotechnological DNA editing tool can be used to human advantage in many ways. In the medical field, CRISPR can be used as a treatment for cancer by addressing particular genetic mutations. It can also improve resistance to diseases and lower health risks associated with genetic factors like heart disease. In the agriculture field, CRISPR can used for crop improvement and edit crop genes to be resistant to pests. In conclusion, because CRISPR allows us to make precise, targeted changes to DNA quickly, and affordably it can be used in many different fields.
Models used to understand CRISPR-Cas9
To understand how CRISPR–Cas9 effectively edits DNA, we used two different ways to understand the process: digital simulation and paper cutouts with instructions. I believe that the easiest way of understanding how this tool works was by the digital simulation. With the digital simulation, I got to see the process in 3D and interactively explore the website. It provided simple definitions of key terms and went into depth when explaining the four main steps in a CRISPR-Cas9 process. A disadvantage of the digital simulation was that I couldn’t get hands-on experience and learn from investigating and collaborating with my group members. The paper cutout activity that we participated in with our group members gave us a chance to get hands-on experience and learn from mistakes and steps. It was also detailed and stated the functions of each tool. A disadvantage that the paper cutout activity had was that it was hard to follow and prone to errors. The paper cutout didn’t give us a chance to see everything in 3D.
I believe that models are a great way to teach students about scientific (or any other subject) ideas. Many individuals take advantage of visual learning. Models are visual, which makes it easier to paint the picture in the student’s minds. Models are also interactive, allowing for hands-on engagement and active learning. Interactive models encourage students to experiment, explore, and observe outcomes in real-time.
https://www.youtube.com/watch?v=jb4xRMyKEz4
https://www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications
https://innovativegenomics.org/crisprpedia/crispr-in-agriculture/