Gene Editing x Stem Cells: The Future of Biology

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Imagine this. One of your loved ones has a serious genetic disease, cystic fibrosis. If this was still 2020, they would only have a 50% chance of living beyond their 40s.

But it’s actually 2040. Like a flip of a switch, scientists are able to replace the gene causing cystic fibrosis, granting your loved one a normal life expectancy. It’s beautiful, it’s amazing, it’s a true feat of science!

But we’re getting carried away. While that is a possibility and likely a certainty in the near future, we’re a long way away from using gene editing clinically on a large scale.

And it’s not just gene editing. Stem cells could save 1.5 million people in the US alone, and that’s a conservative estimate.

To see where these two intersect, we first need to talk about gene editing and stem cells separately. We’ll just cover the basics but trust me, it’ll be fascinating nonetheless!

What Exactly Is Gene Editing?

Gene editing is just what it sounds like: editing your genes. Genes make you you, so being able to edit them is a pretty big deal! There are quite a few ways to edit genes at this point, including Zinc-Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALEN), and CRISPR-Cas-9. They’re all super fascinating and you can learn more about ZFN and TALEN by clicking here, but for today we’re going to zoom in on the fascinating CRISPR system.

CRISPR-Cas9

Unless you’ve been living under a rock or were spending the last few years on Mars, I’m guessing you’ve heard of CRISPR. It’s been getting thrown around everywhere as the one solution to all of our problems.

First off, we need a little history lesson to find out about how CRISPR came about. Don’t worry, this isn’t any old boring lesson. It involves something we’re taught to be very afraid of as kids, bacteria 🦠

Even though we usually think of bacteria and viruses as kind of the same things that want to hurt us, bacteria and viruses have actually been in a fight for billions of years among themseleves. Viruses want to hijack bacteria, just like they do us.

But bacteria thought “not so fast!” and developed a system called CRISPR where they would take the DNA of any hostile viruses and put it into their own DNA so they could recognize the virus later and destroy it.

Now we’re back to the current time, what a ride! So it’s time to dig in: what is CRISPR? For starters, most of the time CRISPR is actually referring to CRISPR-Cas9. But let’s just focus on the CRISPR part for now, which is an acronym.

Now you know why they use so many acronyms in biology! But it’s not as complicated as it sounds. Focusing in on the “Short Palindromic Repeats” part for now, we need to understand what DNA, nucleotides, RNA, and a genome are.

DNA is like the recipe of our cells and also “tells” cells what to do. You may have heard people refer to DNA as a double-stranded helix. It sounds complicated but all it means is that DNA has two strands connected together that are twisted.

Look at that double helix shape; it looks like a twisted ladder! Image courtesy of EDN.

Nucleotides are the building blocks of DNA. If DNA is a recipe, nucleotides are the ingredients. The defining characteristic of these nucleotides is their bases. They’re those letters in the DNA you keep hearing about. A for adenine, T for thymine, C for cytosine, and G for guanine. A pairs with T, C pairs with G. It’s just one of those rules.

The cool thing about DNA is that because of this pairing rule, knowing just what one strand says means you know what the other one says too! Let’s see this in action.

Take a look at the DNA, which is at the top. See how the bases match? Now try and create your own DNA sequence using just that one strand at the bottom. It’s pretty simple!

Turns out the one strand version has a name too, RNA. And it plays a vital role both in CRISPR and in our cells in general. Finally, our genome is the entirety of our DNA!

Now that we have some basics down it’s time we learn about what those “Short Palindromic Repeats” are. Let’s visualize this by using an analogy. Think of the genome as being separated into lego blocks; those blocks are formed by a few different nucleotides. You’re creating a chain of these blocks where you keep repeating the blue block because it’s your favourite colour!

But there are spaces in between those blue blocks, where you place blocks of different colours. Those are your enemies’ favourite colours. You keep them there as reminders of what who you’ve dealt with in the past.

Back to reality now! Those blue blocks are nucleotide repeats. They’re just repeating nucleotides as the name suggests, and they don’t serve a purpose as far as we’re concerned. The other blocks from your enemies are nucleotide spacers. These spacers are where the viral DNA, the DNA of the virus, is stored just like how we talked about during our history lesson. Pretty cool right?

We now understand the “Clustered Regularly Interspaced”! And the “Short Palindromic Repeats” bit is just talking about those nucleotide repeats. This long

This is where we come to the Cas9 part of the name! If the Cas is for CRISPR-Associated and there are several of them, hence the use of numbers after “Cas”. Some Cas proteins are helicases: they unwind the DNA. Some are nucleases: they cut DNA. That’s why CRISPR is so often referred to as molecular scissors.

If the bacterium (singular of bacteria) is injected with viral DNA that it has dealt with before, i.e. it is found as nucleotide spacers in its genome, then it simply creates CRISPR RNA or crRNA. This crRNA comes from that long sequence of repeats and spacers. But instead, it’s a smaller chunk, only containing the needed spacer that matches that viral DNA.

It’s like having a formula sheet during a math test. You can use it to find the right formula to use for a certain question!

A Cas protein then cuts that viral DNA, rendering it useless 🎉 An awesome analogy for this is that the Cas protein is carrying around a mugshot of the viral DNA and as soon as it recognizes it, it’s game over.

But what if it’s new, foreign viral DNA? What if we don’t have a mugshot? That’s where the exciting part comes in; it’s where the Cas proteins get to go chop chop ✂️

In this case, the crRNA can’t act alone, however. It must come together with tracrRNA and then bind to the Cas protein The main role of tracrRNA is to prevent the Cas protein from becoming inactive.

Think of it like this: the Cas protein is the body of a car that holds the passengers and the equipment for doing the work (in this case, cutting viral DNA). The crRNA is the map that helps the passengers direct the car to the right destination. And the tracrRNA is the passenger itself, preventing the car from becoming “inactive” or not actually moving.

Notice the absence of tracrRNA in the inactive Cas protein!

Once the crRNA and tracrRNA come together, the Cas protein take the viral DNA and cuts it apart. Then, it stores the viral DNA in the DNA of the bacterium as a nucleotide spacer.

Now how do we apply this to humans? In this specific case, we use Cas9 proteins. And to simplify the process, the crRNA and tracrRNA were engineered together to form guide RNA or gRNA. So now, our system only has 2 parts

  1. Cas9 protein
  2. gRNA (or sometimes called sgRNA)

When the gRNA finds a match in our own DNA, it uses the Cas9 protein, which acts as scissors and cuts the DNA precisely where we want it to. If we want to not just break a gene but also add a new one, we can add a third part to the mix, some host DNA. That gets inserted in the place of the DNA we previously cut!

The coolest part is that this isn’t just something that’s done in labs! It works in vivo, meaning in actual cells!

If you’re still a little confused, don’t worry, it’s a complex topic! Check out the below video; it’s by far the best one I’ve found that has a balance of simplicity and detail.

It might seem like a waste of time but I highly recommend watching the video twice! There’s a lob to absorb so it really increases your understanding.

What Exactly Are Stem Cells?

Yay, we’ve gotten to one of my favourite types of cells (yes, I have a list of favourite cells)! Think of stem cells as 3-year-old kids who still have aspirations to become an astronaut, painter, and vet all at the same time. They have so much potential and they know it!

But there are varying degrees as to how much “potential”–or what we actually call “potency” in the field–a stem cell has. Those 3-year-olds I was talking about represent the totipotent stem cells. They can turn into any type of cell, including those in the placenta.

Next up we have the pluripotent stem cells. These cells are maybe 8 years old because they’ve ruled out some career options and they can turn into any cell type except for placental ones!

Finally, we have our multipotent stem cells, which are also sometimes called adult stem cells or even somatic stem cells (way too many names!). Think of them as being in high school so they know what subject area they want to explore. Maybe they’re learning more towards the sciences or maybe it’s the arts. They can turn into any cells within a category. For example, skin stem cells can turn into the various cells used to create the skin. But they definitely can’t turn into kidney cells? Or can they? (dun dun dun… we’ll talk more about this later in the article!)

Still don’t think you understand stem cells? Don’t fret, I’ve got you covered. Check out my video that gives you a quick introduction to stem cells!

But why exactly are scientists and the media, so obsessed with stem cells?

It’s because they show promise in so many different areas from organ regeneration to preventing aging! However, like we learned, not all stem cells are created equal. For a lot of those mindblowing applications, we need pluripotent stem cells! And there are only a few types of cells that are pluripotent:

“True” Embryonic Stem Cells (ESCs)

“True” embryonic stem cells come from real human embryos, hence the title “true”. But let’s dig a little deeper into the process. ESCs come from the in vitro fertilization (IVF) process where the egg is fertilized outside of the mother’s body. Once the zygote (just a fancy word for a fertilized egg) reaches a certain stage, it is put back into the body of the mother.

In the case of embryonic stem cells, the zygote must reach a stage called the blastocyst stage and that’s when the ESCs are retrieved.

So by now you might have guessed why ESCs are so controversial. It’s due to much of the same reasons as abortion. Whenever science begins dealing with the components of giving life, ethical debates heighten.

But it’s extremely important to note that these stem cells are only ever used in research if the parents of the embryo have consented to it. According to Dr. Esteban Mazzon, the only blastocysts ever used for research purposes are either poor quality, meaning they would not have been chosen to be placed back into the mother, or have been screened.

Those that are screened are not chosen due to the risk of genetic diseases. However, they are awesome for research since you now have a stem cell with the genetic makeup of a human disease.

Regardless of the controversies surrounding them, ESCs have played a critical role in stem cell research. Those controversies do still exist and are important to address so it’s time we take a look at alternatives!

Induced Pluripotent Stem Cells (iPSCs)

These stem cells actually come from adult stem cells. They are then “turned back” into pluripotent stem cells instead of just multipotent ones using 4 factors called the Yamanaka factors. It’s like you’re turning back time 🤯

So it turns out there actually is a way for kidney cells to be turned into skin cells! You simply use the Yamanaka factors to first turn it into an iPSC which is then turned into a skin cells.

They may seem like the perfect replacement of ESCs since they bypass all of those ethical concerns. Unfortunately, there’s a still a long way to go before we can completely replace ESCs with iPSCs in research.

Somatic Cells Nuclear Transfer Embryonic Stem Cells (NT-ESCs)

These types of stem cells are not talked about nearly as much as ESCs or iPSCs but they have lots of potential as well. They’re both quite similar and very different to iPSCs but let’s first take a look at what they even are.

NT-ESCs come from a process called Somatic Cell Nuclear Transfer or SCNT for short. The goal of the process is to insert the nucleus of a somatic cell into an egg cell and consists of 3 major steps: enucleation, injection/fusion, and activation.

That sounds pretty complicated so let’s break it down together!

  1. Enucleate (or remove the nucleus of) a somatic cell: A somatic cell is a multipotent cell found in the adult body. The enucleation is done so that we can later insert the DNA of this somatic cell into the egg cell.
  2. Enucleate an egg cell: Since we need to put the DNA of our somatic cell in here, we need to get rid of the nucleus of the egg cell. This ensures that the only DNA that is carried forward from here is the somatic cell’s and not the egg cell’s.
  3. Insert the nucleus of the somatic cell into the egg cell: To understand this part, we need to cover a bit more basic cell biology but I’ll make sure to keep it super simple! Most cells in our bodies have 46 chromosomes in the nucleus. Chromo-whats? If you think of DNA as the recipe to our cells then chromosomes are just tightly wound up recipes. 23 come from our biological mother and 23 from our biological father. But there’s an exception to that 46 rule when we come to our gametes, the egg and sperm cells. Those only have 23 chromosomes. So in order for a egg to be fertilized, it must have 46 chromosomes. Inserting the nucleus of a somatic cell, which contains 46 chromosomes, “tricks” the cell into being fertilized. How clever!
  4. Activate the zygote: This step might seem strange. The egg is fertilized, what do we have to activate? What we’re not often told about is that through a process we won’t get into right now, the sperm causes changes in the levels of calcium in the egg cell that activate it and kick it into the cell cycles, where it starts to multiply. It’s almost like the on button on a remote controller. All of those amazing Netflix TV shows are waiting for you. But until you turn on the TV by pressing a button, nothing will happen. But since there’s no sperm entering an egg cell during SCNT, we need to simulate that another way. We can do that both using electric simulation and chemical simulation. This is like bypassing having to turn the TV on and getting your sibling to do it instead!
Inspired by slides from Dr. Esteban Mazzoni

And now the process can take two paths and where the remainder of the steps diverge. We can either use these zygotes for reproductive cloning or therapeutic cloning.

Watch this video to get a quick summary of what we just went over if it’s still unclear!

Meet Dolly the sheep, a cloned animal! Image courtesy of John Chadwick — AP/REX/Shutterstock.com

Reproductive cloning is where the zygote is put into the uterus of a surrogate mother and is then given birth to normally. And yes, it’s exactly what is sounds like. It’s cloning! You get a complete genetic clone of whatever organism’s somatic cell nucleus was used. Of course, this hasn’t been done for humans before but it has been done on mammals! Look no further than Dolly the sheep 🐑

Therapeutic cloning is what we’re interested in and it has been done for humans before. This follows the exact same process but instead of being placed in a surrogate mother and allowed to fully develop, the zygote is only allowed to develop into a blastocyst, at which point embryonic stem cells are derived from it. This step is super similar to ESCs from above, isn’t it?

This process is so fascinating so make sure to follow me and watch out for a full article on SCNT and NT-ESCs coming in the near future!

Now, remember how I said iPSCs were lacking in several areas? Even though they’ve been hogging a lot of the attention, several studies show that NT-ESCs are actually more similar to “true” ESCs derived from the IVF procedure, which is often called the “gold standard” in the field.

There’s so much more to these fascinating NT-ESCs that we can’t get into right now but be sure to watch out for an entire article coming out soon on this topic 👀

Parthenogenesis Embryonic Stem Cells (pESCs)

Are you kidding me? There’s another way we can derive embryonic stem cells? Yes, isn’t is amazing? Parthenogenesis is where a zygote, a fertilized egg, is formed without the use of a sperm. It’s used by some invertebrates as a form of asexual reproduction.

But that’s not how humans work, right? Correct… that’s not how our processes work naturally and embryos created in that way actually won’t develop into a fetus but they can become blastocysts and that’s when we can derive ESCs from them.

So how the heck does this work? It’s super similar to the activation of the egg cells we talked about in the previous section. But here, since there aren’t 46 chromosomes, the embryo can only get so far! However, it’s perfectly far enough for our purposes of getting ESCs.

Another really interesting thing about pESCs is that they almost completely bypass the ethical debates that are happen around “true” ESCs. They can even be applied to NT-ESCs since they have the potential to develop into an embryo and then a human. However, since pESCs are not viable enough to become actual babies, that discussion is not necessary💡

Where The Two Meet In the Middle

Now we come to the most epic cross over since Avengers: Endgame! There are so many ways in which gene editing an revolutionize stem cell science and we’re going to learn about a few.

Differentiation: The Art of Choosing

First off is for differentiation. We haven’t talked about this yet but it’s the process by which totipotent cells become pluripotent and then multipotent. Going back to our career analogy, differentiation is similar to choosing a career path.

Dr. Esteban Mazzoni, professor of Biology at New York University, explains this really well in one of his lectures

At every step of the way, the cells have to choose a path to become something at the expense of the possibility of becoming something else. [And each time the cell makes a decision] by definition, [it is getting] farther [away from] any other place that [it] could have gone.

This is great and all but what does gene editing have to do with it? iPSCs are great for research but they can be difficult to guide along a specific differentiation path, like getting them to become skin cells.

The process of differentiation!

By harnessing gene editing, we can simplify this process which allows us to study diseases without having to harm actual humans! It could even be used to generate artificial organs.

It’s good to note that the success rate of editing iPSCs hasn’t been exceptionally high but great work is being done in the field.

Studying Degenerative Diseases

Another amazing use of gene editing and stem cells is for studying degenerative diseases. It’s kind of a follow-up to the last point but let’s dig into some specific examples.

One instance is sickle cell disease, an inherited disease where there aren’t enough healthy red blood cells in the body. Researchers corrected the mutation that causes the disease in hematopoietic stem cells, just a fancy word for blood stem cells. When the hematopoietic stem cells differentiated into the specific type of red blood cells, called erythrocytes, they showed decreased signs of sickle cell disease!

This is what sickle cells look like; they’re in a crescent shape and that decreases their function. Image courtesy of Get Healthy Stay Healthy.

Another example comes from work with multiple-system atrophy, a neurodegenerative disease that affects your body’s body’s involuntary functions like breathing and blood pressure. By investigating the disease using CRISPR in human neurons that were derived from iPSCs, researchers found that a reduction in coenzyme Q10, a type of coenzyme (a compound that helps an enzyme function), leads to neuron apoptosis. That means neuron death! This will help pave the way forward for treatments to this rare disease!

There are so many other examples that I won’t cover right now, but you can clearly see how both common and rare degenerative disease can be studied and possibly cured using gene editing and stem cells.

Identifying Important Genes

The last fascinating application we’ll talk about today is identifying important genes! Stem cells have opened so many doors for studying cell pluripotency and cancer. By using CRISPR to “knock-out” genes, researchers have been able to identify specific ones that contribute the most.

It’s a bit of a brute-force method, almost like punching someone in the face many times to see which of their teeth has the strongest root (which I do not recommend by the way). But it’s a great way to study genes anyways!

Challenges Ahead

While this technology holds a lot of promise, there’s still tons of challenges we have to face too.

One of those is off-target mutations and effects. This is more so related to CRISPR than stem cells but it is when gene editing has some unexpected effects in an unintended area of the DNA or the body. Some studies suggest that off-target effects may be a result of bad gRNA and not the process itself, but it’s still one of the biggest hurdles in the field.

Another one is the use of CRISPR and stem cells in vivo, meaning inside the body instead of in a lab. Some treatments can be done by collecting stem cells, editing them, and then inserting them into the body but others must be done on the organs in vivo. Currently, there are challenges with not being able to turn off the CRISPR system, which means the risk for off-target effects skyrockets. There’s research being done here too, but it’s something we need to be aware of in the future.

The last challenge I’ll address here is immunity against the Cas9 protein. Cas9 was actually found in Streptococcus pyogenes, a type of bacteria that can often infect humans. That means we may have immunity against it and while that’s good if the body was actually under attack, it means we may have problems getting CRISPR to work without our own body attacking it.

And you’ve reached the end! I hope you learned a thing or two about stem cells, gene editing, and their intersections!

References

For a list of my references, click here!

Hey there 👋 Parmin here; I’m a 15 y/o student studying stem cells at The Knowledge Society 🧪 Everyday, I aspire to uncover the secrets of biology and learn something new! Make sure to follow me on Medium to hear about every new article I post, connect with me on LinkedIn, or contact me at parminsedigh@gmail.com! Also subscribe to my monthly newsletter to learn about every cool, new thing I’m working on ✍️

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Parmin Sedigh

Parmin Sedigh

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Science communicator trying to learn something new everyday | Published in Start It Up, Predict & The Writing Cooperative