Simulating genetically modifying a defective cancer detection protein with CRISPR
So I came across an app called Benchling, and it seemed to be extremely cool. I’m interested in Gene Editing and specifically CRISPR. Since I’m in 8th Grade I don’t really have the opportunity to get hands on with this type of stuff so online simulation was the perfect alternative for me. I decided to target defective cancer detection proteins, more specifically p53.
So why did I pick p53?
10% of all Cancer is inherited by faulty genes. 10 out of 100 people lost the genetic lottery and have to deal with an nigh uncurable terminal illness. Some of the most common cancers like Breast Cancer and Prostate Cancer come from this faulty genetic suppression. TP53 is an important genetic cancer suppression protein and is one of the most commonly mutable ones.
How does p53 even work?
Imagine p53 as the chief security officer in your body’s control center, the nucleus of your cells. Its main job is to keep a watchful eye on the DNA, the blueprint of life, making sure everything is in order. It’s like a superhero ensuring the peace and harmony of the cellular city.
When there’s a disturbance, like damage to the DNA caused by factors such as sunlight or smoking, p53 springs into action. It’s a bit like an emergency response team activated to handle the crisis. P53 assesses the situation and decides on the best course of action to maintain cellular health.
Rather than using force, p53 acts more like a wise leader. It enters the nucleus and starts giving orders to specific genes, directing them to execute a plan. One of its key strategies is to hit the brakes on the cell’s normal activities, like a traffic cop temporarily stopping the flow. This pause gives the cell time to fix any damage to the DNA — a bit like a repair crew fixing potholes before traffic resumes.
However, if the DNA damage is too severe and beyond repair, p53 makes a tough call. It initiates a process called apoptosis, which is like a self-destruct mechanism for the cell. This controlled cell death ensures that the damaged cell doesn’t cause harm to nearby cells, preventing potential trouble.
P53 also has a retirement plan for cells. If they’ve been through a lot and could potentially cause issues, p53 can put them into a state called senescence. It’s like telling old soldiers to hang up their uniforms — they won’t be dividing and causing problems anymore.
While p53 is usually a superhero, ensuring that damaged cells don’t turn into bigger issues like tumors, sometimes it faces challenges. If it’s not working properly due to mutations, it might miss the warning signs, allowing damaged cells to grow into tumors. This is when problems like cancer can emerge. So, in the grand scheme of things, p53 is the guardian of cellular order, but when it falters, it can lead to cellular chaos.
So I started to look for defective plasmid models online, I found one with a flagged mutation of changed arginine 248 to tryptophan. This is an extremely common mutation that can happen through an variety of factors. While this specific mutation is what we used the method can be widely transferred to any other mutation as long as it is within the p53 protein
Now I had to find a normal non-faulty p53 plasmid which was surprisingly difficult. However after I found it the task seemed pretty simple, cut the defective parts of the cell and paste in the healthy part to make a new plasmid.
The hard part was actually inserting this new plasmid into the gene, which is TP53. This gene is located in Chromosome 17 and using Benchling’s CRISPR feature I can target this protein and add it via insertion.
What is CRISPR?
CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is a revolutionary genome editing tool that has transformed the landscape of biological research. At its core, CRISPR operates through a two-component system: guide RNA (gRNA) and the Cas9 protein. Scientists design a synthetic gRNA that precisely matches the target DNA sequence within a cell or organism.
This gRNA serves as a molecular guide, directing the Cas9 enzyme to the specific location on the DNA where changes are desired. Once the Cas9 protein reaches its target, it acts as molecular scissors, creating a precise cut at the designated site. This cut triggers the cell’s natural repair mechanisms, which can be exploited to introduce desired modifications to the DNA sequence.
So since we know where TP53 is (Chromosome 17) we now have to figure out which exons (An exon is a segment of DNA in a gene that codes for the formation of a functional RNA molecule, such as messenger RNA (mRNA). Exons are transcribed and retained in the final processed RNA, which is then translated into a protein or functions directly as a functional RNA molecule.) are Hotspots for Mutation!
After researching the exon hotspots for mutation I found that it is the exons 4–9 that are extremely prominent in cancer mutations, so I decided to target these exons and ignore the rest.
Benchling’s CRISPR function automatically does On-Target and Off-Target scores for you (On-target score refers to the efficiency and accuracy of CRISPR-Cas9 in precisely targeting and modifying the intended DNA sequence. Off-target score measures the potential for unintended or undesired modifications to occur at locations other than the targeted site during CRISPR-Cas9 gene editing.) So we want the highest On-Target score for the highest accuracy and smallest chance of bad side affects.
After careful picking I narrowed it down to these few cut positions.
As you can see all of these positions have a score of at least 70, this ensures a decent amount of accuracy.
Someone could actually do this in real life! Through CRISPR testing kits you could basically make your own CRISPR. It’s really crazy to think that a few years ago Gene editing had an extreme high barrier to entry but now a kid can simulate Genetic Modifications in a few hours!
Here are some really cool videos that helped me understand what was happening in Benchling, I really recommend you watch these as Gene Editing becomes extremely interesting once you get into it.
