Gene Therapy To Solve Cystic Fibrosis

Aryan Khimani
8 min readJan 1, 2022

What if I told you that you can only live till you are 40, and till the day you die you’re going to be in constant pain, the ER is going to be your second home and at any moment your body could just quit on you. How would that make you feel? For me, it would make me feel crushed, it would make me feel awful and most of all I would feel a strong sense of injustice because I couldn’t control this fate.

This is the life of people who have Cystic Fibrosis. Modern medicine has definitely improved the lifespan of those suffering from Cystic Fibrosis (CF). The lifespan of a person with CF went from the age of 8 in the 1900s to 45 here in Canada (30 in the US). That is a massive improvement, but it still doesn’t change the fact that the average is still pretty low when compared to an unaffected person. This is why I propose we use an emerging technology called CRISPR as a form of gene therapy for those suffering from Cystic Fibrosis. To prove it would work I’m going to show you my research and how the system would work. Before that, you need to know more about Cystic Fibrosis.

Cystic Fibrosis

People Affected by Cystic Fibrosis

It mainly affects the lungs

Cystic Fibrosis is a single gene mutation in the CFTR gene on chromosome 7. This disease is an autosomal recessive disease which means it has to be inherited from both parents to be an issue and if one allele is inherited they would be a carrier of the mutation. Within Canada 1 in 3600 are born with Cystic Fibrosis, while in America, though the number of babies born with Cystic Fibrosis each year in the United States is 2,500 however, USA’s current total of people with Cystic Fibrosis is 30,000 and more than 10 million people don’t even know they are carriers of the disease. The types of CF are broken into 6 different classes! Scientists estimate that there are over 1,000 genetic variations in the CFTR gene, but one of the most common ones is ΔF508, which stands for the deletion of three bases pairs of phenylalanine at residue 508.

The daily life of people with Cystic Fibrosis

Cystic Fibrosis is a gene mutation that mainly affects the cells that produce mucus. The organs that get hit the hardest are the lungs and digestive system. This is due to people with CF making sticky and thick mucus. Which at first, might not seem like that big of a deal but for the body, it poses the threat of creating clots as well as plugging up your lungs. In the lungs, there is a layer of mucus normally that protects the lining of the airways stopping bacteria and foreign particles that you breathe into your lungs that would otherwise cause an infection.

A good diagram to show what happens if you have Cystic Fibrosis

To get the trapped bacteria and particles out of your lungs the Cilla moves it along and out, but when someone has Cystic Fibrosis the mucus becomes more viscous making it too much for Cilla to do its job. This is because Cystic Fibrosis codes for a membrane protein or gateway protein that allows the movement of chloride and sodium into the mucus and back into the cell making the mucus less viscous. However, due to the mutation of the CFTR gene the protein is folded incorrectly so it doesn’t get outside of the cell stopping the passage of chloride and sodium into the mucus. Creating an imbalance that sucks the water out of the mucus through the process of osmosis and slowly over time, the mucus builds up and eventually clots the airways.


These are some extra symptoms

Your symptoms are displayed from the moment the CF patient takes their first breath. As a baby, many symptoms like Meconium Ileus, a bowel obstruction, are life-threatening and a surgical emergency, these symptoms could lead to organ failure and even death. For children, a big concern is their pancreas mucus stopping the passage of enzymes and vitamins that the body needs. While in adulthood, symptoms get even worse as the threat is constant with common symptoms like lung infections, Pancreatitis, Pneumonia and Arthritis. Of course, there are treatments to reduce the effects, but not a cure for many.

Overview of Cas9

Before we talk about my proposed cure let’s talk about how Cas9 works. Cas9 or CRISPR can edit the genome of any living thing, similar to how Ctrl+F and Delete works on a computer. However, Cas9 isn’t just one protein as there are many variations of this protein varying in size, PAM and bacteria of origin. For example, we are going to look at the most common one which is found in Streptococcus pyogenes. Cas9 is at the end of the day a protein meaning it doesn’t have eyes. So for Cas9 to be able to find the target sequence we need to give it a guide or in this case a guide RNA (gRNA).

Most gRNAs have three main features the first two are crRNA and tracrRNA which the lab turns into a Single Guide RNA (sgRNA). The third feature is in the gRNA sequence itself, it is a specific extension at the end of the gRNA which we call the PAM. It has a 2–6 base-pairs length that needs to match with the DNA or else the Cas9 Protein won’t make a Double-Strand Break (DSB) required for its function

The CRISPR I Chose

As I said in the paragraph above there are many different types of Cas proteins that could be found in various bacterias, have different PAM sequences and efficiency rates. I choose Cas12a or Cpf1 for a multitude of reasons. One of the reasons being Cas12a was smaller in size when compared to Cas9 as well as Cpf1 only needs the crRNA to work instead of crRNA and tracrRNA. Cutting the amount of genetic data needed as Cpf1 is approximately 3.8kb while Cas9 is approximately 4.1kb. Another reason is that the PAM sequence of Cpf1 is filled with Thymine such as TTTV, TTTN,TYCV and TATV making these PAM sequences a better fit for this type of edit as it allows it to be closer to the target sequence without lengthening gRNA above the norm. Lastly, the Cpf1 score for off-target effects is superior to Cas9, which in my opinion is most important as mistakes in the lung airways could potentially worsen the condition for CF patients.

Gene Delivery Methods

There are two types of Gene delivery methods In Vivo and Ex Vivo. In Vivo is when you inject the cure into the person while Ex Vivo is when the target cells are taken outside of the body and edited before they are put back into the body. Ex Vivo method of gene therapy cannot be used to treat Cystic Fibrosis as we can’t simply take the cells we require from the lung’s airways, edit them and place them back in the lungs. Therefore, In Vivo is the best way for us to deliver Cpf1 to the target destination. However, we can’t just inject Cpf1 into the body hence, we need to choose a vector to pass the protein to the target cell region. That is where AAVs come in.

Adeno-associated virus (AAV)

AAV or Adeno-associated virus is the perfect method to deliver Cpf1 to the target cell region but aren’t we actively trying to stay away from viruses much less willingly injecting it into our body?

This isn’t your traditional virus as it has very low pathogenicity and low toxicity meaning it doesn’t cause much damage to the body. It only provokes a very low immune response but in addition to that, it can infect both dividing and non-dividing cells, and most of all countless papers testing its efficiency and safety. Making it the golden standard for gene therapy delivery but like the Cas protein, there isn’t just one kind of protein, AAV has many different variations from AAV1 to AAV9. Of them, AAV6 is best for this type of gene therapy.


AAV Diffusion in Spontaneously Expectorated CF Check out the link: Sputum

This variation, in particular, is the best one for the job because of two main reasons. One of the big reasons is its tropism. Tropism is the virus’s ability to productively infect a particular cell and for AAV6 it’s the heart, liver and most importantly airway epithelial cells. Another reason that AAV6 is the best one for the job is that it’s the most efficient at penetrating the Mucus Barrier. That is where many treatments fail because it’s very difficult to cross the mucus barrier as its main function is to stop bacteria and other particles from penetrating the lungs and the cells around it. Especially with Cystic Fibrosis, the viscous mucus barrier hinders the Gene delivery methods from reaching the intended target region. When comparing other methods of delivery AAV6 vector provides significant advantages over other AAV vectors for lung gene therapy.

Final Project

Therefore, by combining Cpf1 and AAV6 into one cure you can theoretically solve Cystic Fibrosis. This would require the plasmid for Cpf1 to be injected in AAV6 for delivery into the target cell to make the necessary genetic modification. Previous clinical studies conducted for solving other monogenic mutations using CRISPR-Cas9 have shown promise, in particular, Sickel Cell Diesae and β Thalassemia human trials. Curing Cystic Fibrosis won’t just rid of a serious genetic disorder but will show the world that Gene Editing Technologies like CRISPR are here and ready to be implemented ushering in the new era of the Genetic Revolution.

Cool Resources and Citation 😎:



Aryan Khimani

I’m 16-years old who is very passionate about using Gene Editing and Machine Learning to disrupt the world of biomedicine, using my knowledge of Python, AI, ML