CRISPR in a nutshell
An easy Guide for anyone to understand CRISPR
Your DNA contains all the necessary information to make you who you are. Nowadays, there is a technology that is easy and cheap enough to biohack yourself however you want and this way, prevent or cure diseases. But it could also create a “superior class” of human beings. So the real question isn’t could we, but “should we?”
A brief story
In our first attempts to edit our genome, cells were exposed to radiation and it was expected that random mutations in the DNA would happen. Then, we were able to insert a DNA sequence at a random location. We had to go through technologies like ZFN, or TALEN, but it wasn’t until 1987 that scientists found a genetic engineering tool that could be as precise, affordable and easy to use as possible: CRISPR.
It is worth mentioning that CRISPR is nowadays used as a genetic engineering tool that could be considered quite modern. Yet, it hasn’t been always like that…
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Despite what you may be thinking, the history of CRISPR goes back to even millions of years ago! Yes, this marvelous breakthrough hasn’t been ‘invented’ by the humankind, but rather by the millions of years of evolution of bacteria’s immune system against viruses.
Viruses are organisms that cannot reproduce by themselves. On the contrary, they need a host to ‘hack’ their system by ‘injecting’ their DNA into the cell, and that way the cell will create more viruses. Of course no cell would ever like this to happen. Thus, they have naturally evolved to have a ‘system’ that protects them against the hackers (viruses) which scientists have now called CRISPR 🙌.
How does it work?
Going straight to the point, if you look “CRISPR” up in Google probably some of the first words that you’ll find will be “Clustered Regularly Interspaced Short Palindromic Repeats” 🤨 (obviously these words didn’t make any sense to a high school student like me at that point).
Because of that, I started to read lots of articles, watch lots of videos, talk to experts and after some weeks, I came up with which I hope is a much easier way to understand CRISPR and its main components:
📑 CRISPR: an archive of ‘samples’ of genes that bacteria take from the invader. This way, the next time the invader is identified, the bacteria will activate Cas9 and cut those specific sequences, leaving the virus inactive.
✂️ Cas9: protein (molecular scissors ) that can cut specific sequences of DNA with the help of a gRNA.
📱 gRNA (guide RNA/GPS): a sequence of RNA that works together with Cas9 and tells it where to cut. It starts with a sequence called PAM.
🏷 PAM: a short sequence of normally 2–6 base pairs of DNA that identify (tag) a specific sequence. This is the only way that bacteria can distinguish between the sequence that is the bacteria’s memory and the one that is in the invader. In the laboratory, PAM shouldn't be present in the gRNA so Cas9 doesn’t cut it.
🧩 Template DNA (replacing part): this component can be optional. Meaning, it’s only going to be used if we are doing a knock-in of a gene.
*Off-target effect: CRISPR cuts somewhere that is not the ‘target’/expected place. In other words, an error ⚠️.
Other Cas enzymes
Although Cas9 is the most widely used enzyme for CRISPR for being the most effective one, there are other types of Cas. Here are some of them:
🦠 Cas-3: bacteria use it naturally to degrade/shred up foreign DNA. Scientists are now using it to kill antibiotic-resistant bacteria. Its gRNA can be engineered to target a specific sequence and this complex is also called Cascade.
✨ Cas-13a: it can be used to reduce the expression of a gene (knockdown)without having to leave it completely inactive. Scientist Feng Zhang has used an inactive form of this enzyme ‘combining’ it with GPF (Green Fluorescent Protein) to track cells that have been infected by a virus like Zika or dengue.
📅 Cas-13b: it targets RNA, so its effects are temporary, but Zhang’s team suggests it could be used to ‘modify’ proteins in the pathogenic stage to slow down the disease progression. It has been used by Zhang’s group to ‘correct’ the mRNA of patients with Fanconi anemia.
Ways of using CRISPR-Cas9
As you may imagine, the real breakthrough for this technology hasn’t been the simple fact that it exists in bacteria. We had to find a way to make this work for other living things, including us. Therefore, the scientific community has been doing research and it’s been found that there are are several ways in which CRISPR can be tweaked to adapt it to the purpose of each experiment.
Here are some of how this gene editing tool can be utilized to modify in some way an organism’s genome:
😵 Knockout: permanent deactivation of a gene by cutting it. After the cut, the cell will undergo a process called Non-Homologous End Joining (NHEJ) which will try to repair the cut made and there is a chance that due to the changes made, the sequence won’t be the same and as a result, there won’t be a protein or the protein produced won’t work.
The way I’d apply this technique is to knock out a gene responsible for a disease such as cancer. For example, oncogenes play an important role in this disease, and perhaps by knocking them out, we’d be able to slow down or stop the uncontrollable cell growth that occurs in cancer.
📥 Knock-in: the opposite of a knockout. The goal here is to insert a DNA sequence into the cell’s genome. This is the kind of technique in which a template DNA could be used. An example of this is an experiment in which our goal is to make an organism glow in the dark. For instance, the yeast wouldn’t normally have a gene that codes for a fluorescent protein 🥴. However, we can make that happen by doing a knock-in of a gene called GFP (Green Fluorescent Protein).
✅ CRISPRa (CRISPR activation): using a variant called dCas9 (dead Cas9) with a transcriptional activator, this complex will only bind to the DNA sequence and ‘tell’ the cell to transcribe the desired gene.
This technique could have either an advantage or a disadvantage, depending on what the purpose of the experiment is. If we are willing to make a more lasting and permanent change, maybe this isn’t the most promising option. But, if we don’t want to ‘mess up’ with an organism’s genome, this could be the best option.
❌ CRISPRi (CRISPR interference): same dCas9, used with a transcriptional inhibitor/repressor. Its advantages or disadvantages are the same as in CRISPRa but its effects are the opposite.
This essentially refers to how we are going to get the CRISPR complex into the cells. The method we use depends on whether we want the gRNA and Cas to stay in the cell permanently (stable) or not (transient). Transfection or delivery methods are also classified into chemical, physical and viral. Next I’ll be explaining how each of them works and their main pros and cons.
🍟 Lipofection: chemical and transient method in which the CRISPR complex is ‘wrapped’ by lipids which facilitate the entrance to the cell by *endocytosis. It is cost-effective but less efficient.
⚡️Electroporation/nucleofection: physical and transient method that applies an electric pulse to the cells to form pores in their ‘envelope’ (plasma membrane) for CRISPR to enter. This method is easy to do, efficient and fast but it requires special equipment
💉 Microinjection: physical and transient that uses a microscope to see the targe and a microneedle to inject CRISPR. It’s commonly used to genetically modify embryos. It can be efficient if performed correctly.
🦠 Virus: viral and stable method. They’re compatible with many types of cells and are highly efficient. Some of the viruses used are lentivirus, adenovirus, adeno-associated virus, and herpes viruses. Some of the main differences between those is how ‘dangerous’ they can be for the target cell and how much DNA they can carry inside them.
From my perspective, CRISPR is all about solving problems. Think about some of the world’s most important problems regarding health. Did cancercome to your mind? According to the WHO, cancer is the second leading cause of death globally, and it was responsible for an estimated 9.6 million deaths in 2018.
If you were born in a time when this and more diseases were pretty much incurable or even considered a death sentence, I understand that it might be incredibly mind-blowing to think that now, thanks to advancements like CRISPR and other cutting-edge technologies, we can have hope in innovative solutions, but I’ll now explain how this is possible.
Even though there already exist treatments, such as chemotherapy or radiation. They have many drawbacks, including the side-effects, which are a consequence of their lack of effectiveness to kill cancerous cells only. Not to mention that I’m almost sure that you have heard that there are patients who are told that treatments like this are not suitable for them, which is evidently a great problem.
As mentioned above, the lack of effectiveness had been one of the main problems of previous treatments for this disease until now. Fortunately, scientists have probably come up with a solution for this.
Its name is CAR T-cell Therapy. The main idea here is to extract the patient’s blood and genetically modify to ‘enhance’ their immune system so it can fight cancerous cells in a tougher and more effective way.
To illustrate this, we can imagine how all of these happens at the cellular level.
First, we need to remember that T-cells are types of immune cells. And there are also many types of T-cells, including cytotoxic, helper, regulatory, natural killer, and memory T-cells. These have different functions like distinguishing infected or cancerous cells 👀, recognizing antigens 🦠 or activating ⚠️ macrophages (other white blood cells).
T-cells are able to recognize cancerous cells because they have a TCR (T Cell Receptor). This TCR interacts with a complex in other cell’s surface called HLA (Human Leukocyte Antigen). An antigen is normally a structure in the surface of pathogens that makes our immune system react. However, in this case, the HLA is a type of antigen that’s present in most of our cells and displays samples of proteins that are being made inside the cell to T-cells so they can decide whether the cell is cancerous, has been ‘hacked’ by a virus or is completely healthy and isn’t a threat for the rest of the organism.
You can also think of the TCR and HLA interaction as when you’re at the airport (a living organism) and the security staff (T-cells) tell you (HLA) to show them what’s inside your backpack (cell) and they do this as a protocol to keep everyone else (other cells) safe (healthy). See why this is important? 😉
Going back to CAR T-cell Therapy, it can now be understood that if T-cells are genetically modified to express a CAR (Chimeric Antigen Receptor), they will be able to identify 🔍 cancerous cells from healthy ones in a much easier way, being a more effective treatment than chemotherapy.
Ethical concerns and why you should be informed
As you may imagine, the possibilities for technologies like this one are almost endless. We can begin by talking about curing blindness but we should also consider the possibility to create ‘designer babies’ and ‘superhumans’. And by no means, I am trying to say this is bad. On the contrary, I am trying to communicate the tremendous relevance that this tool is going to have in the near future. But don’t take my word for it, take Yuval Noah Harari’s, who mentions in his book “21 Lessons for the XXI Century”:
Biotechnology and Artificial Intelligence (AI) could divide humanity into two groups: superhumans and the useless class.
The picture above shows one of my favorite quotes ever. Which brings me to the next point which is: are we playing god by doing genetic engineering? 😧
From my point of view, we could also be playing God by doing any other medical treatment. Yet, I think that what is truly causing a lot of controversy is the fact that gene editing could be used to make superior people.
Right now, we are all aware that there are different social classes in the world, which follow the rules of the economic system. Nevertheless, if we created ‘designer babies’ and gave them special ‘features’ such as a higher IQ, extra strength or a high lifespan, we wouldn’t only need to worry about whether someone owns one dollar or a million, but also about whether someone has all these abilities or not. The gap would between each social class could increase due to all the advantages that a group of people could have.
Now, you may be asking yourselves: why could this be so bad if as in any other medical advance people will eventually have access to it? 🤔
Well, that’s actually the core question. If we can ‘democratize’ this and many other new and relevant technologies, we wouldn’t need to worry about the gap. Superhumans wouldn’t exist because we could all enjoy the advantages that genetic engineering can give us as humans. In other words, we would all be superhumans. It wouldn’t be a ‘luxury’ of a small group of people, but a right.
Going back to the relation of genetic engineering with health aspects, I do believe that we should be careful, enough research should be made before we can try this in human beings and we should keep in mind corresponding regulations. Nevertheless, we should also put hands into work NOW. CRISPR already exists, it’s now in our hands to use it wisely for everyone's benefit. Diseases which impact one in every thousand people and others which impact millions, can now be treated thanks to gene editing. Will we let God play as long as we don’t use genetic engineering in humans to cure diseases?
Whichever your point of view is on this topic, the undeniable fact is that this technology could actually be quite important soon. Thus, I think we would all rather be at least a little bit informed than just rebut these new ideas. By reading this article you’ll have learnt the basics of how CRISPR, a gene editing tool, works.
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