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Before jumping into CRISPR technology, we must learn how researchers cut DNA through gene editing tools. How do these tools differ from each other? 

The chances are that you have heard of the advances in gene editing. In case you missed it, scientists can alter the sequence of mammalian genes (and other organisms) through a relatively new technology called CRISPR-Cas.

This highly efficient tool has gained popularity not only within the research community but also in the non-scientific community. Why? It’s easy to use, takes little learning time, and is cost-effective. Most importantly, it can alter the genes and gene expression of cells in the culture dish and in living multi-cellular organisms. These properties have led to promises and speculations about the future, think disease-resistant crops and disease prevention or treatments. And then, we see certain enthusiasm among the part of the fitness community that wishes to increase sports performances.

If CRISPR is brand new to you, there’s no need to worry; we’ve got you covered in this post. But before storming into the CRISPR field, it’s best to know your gene editing. This post and a couple of future ones will cover the following topics:

⊕ Genome editing: the tools leading up to CRISPR and how they work.

⊕ What CRISPR is and how it works.

⊕ How labs use different types of CRISPR systems.

⊕ Treating diseases with CRISPR.

⊕ Proposed uses of CRISPR (for example, manipulating human embryos).

For now, let’s start with gene editing, though.

The genome editing tools leading up to CRISPR and how they work

What you need to know:

⊕ We can alter genes and gene expression very specifically with genome-editing tools

⊕ There are different ways or technologies to edit genes

⊕ CRISPR is the latest and most user-friendly tool for this purpose. It can be used to delete or add nucleotide or larger sequences, taking advantage of the cell’s DNA  damage repair system

⊕ There are many applications to the tool, and we’re just seeing the beginning of it

DNA can be inserted, deleted, or replaced in the culture dish and in living multi-cellular organisms. This is genome engineering.

While we’re here: Is gene editing and genome engineering the same thing?

These two terms are often interchanged. The simple answer, however, is “no”. While all genome editing tools are nuclease based (more on this soon), genome engineering is more general. So, genome editing tools are part of genome engineering. The opposite is not necessarily true.

The DNA damage repair

Before going into how genome engineering or genome editing works, it is wise to understand the DNA repair mechanisms intrinsic in all of our cells. We will get to CRISPR soon, that’s a promise, but we first need to know this:

DNA is constantly challenged with stresses from outside the body (think sun or smoke) or inside the body (think metabolism). To tackle these challenges, the cells have developed a repair mechanism that can repair these breaks. We call this DNA damage repair. Improperly repaired DNA breaks can lead to the accumulation of thousands of DNA lesions per day. This could eventually lead to one of the following outcomes:

Senescence – The cell gets into an irreversible cell state where it stops dividing. It gives up.

Apoptosis – Programmed cell death. The cell decides it’s not worth it anymore and starts signaling to pull the plug.

Cancer – Uncontrolled division of cells. In the context of DNA damage, DNA breaks can lead to overall gene instability and the possibility for the cell to transform into a cancer cell.

The first two outcomes are part of the cell’s response to save it from the third one: cancer. As we know, cancer means big trouble, and we want to avoid this by all means. That’s why we have an efficient DNA damage repair system that protects our DNA when needed.

The DNA damage repair pathway can fix a break in different ways

Let’s simplify this a bit to understand the outcomes of DNA damage repair better. Imagine a double-stranded break caused by irradiation.

Several responses are possible:

1) The double-strand break is “glued” together properly, and it looks normal in the end:

2) The double-strand break is fixed by using the sister chromatid as a template after the DNA has been replicated:

3) Errors can occur in the DNA damage repair, leading to small nucleotide insertions or deletions around the broken area. This is done by adding nucleotide(s) to the sequence or by “chewing” nucleotide(s) from the sequence: 

Why do we need to know this? The answer is that genome editing relies on the cell’s DNA damage repair system. You will see.

There is a lot of interesting information about DNA damage and related topics. With this brief introduction, we think you are ready to understand genome editing and, eventually, CRISPR.

Drumroll!

How to make changes in the DNA sequence using gene editing: 

This is easy! We cut the DNA with a pair of scissors at a sequence-specific site. From this point, we rely on the cell’s repair machinery to do what it’s intended to do: fix the DNA. This results in one of the three outcomes shown above.

In this example, the pair of scissors represent the main tool in genome editing, which is the endonuclease. It is an enzyme that cuts the DNA to form DNA breaks.

In the lab, we can direct these nucleases to specific locations in the DNA by linking them to some sort of DNA-binding module. The module type depends on the genome editing tool we use to cut our “gene of interest” or “sequence of interest”.

Of course, in cases where we want to change the gene sequence and cause insertions or deletions, we rely on the repair machinery to do a bad job.

What happens when we cut DNA, and why do we even want to do it? 

As researchers, we can design the DNA-recognising or DNA-binding module to target a specific DNA sequence. How we do that depends on the tool we will use (coming up soon).

Once the DNA-recognising module has guided the endonuclease to the target sequence, the nuclease can cut the DNA. In doing so, it will trigger the activation of the DNA damage repair system of the cell to fix the cut.

Relying on the errors of the DNA damage repair to cause mutations in the gene

Remember that the DNA repair machinery could cause errors upon double-stranded breaks? Changes like these are actual mutations and can cause abnormal gene expression if introduced to a protein-coding gene. This can result in a non-functional or absent protein (also called a knockout). In other words, by using genome editing tools, we can direct mutations to genes we want to be knocked out.

Relying on the error-free DNA damage repair to insert sequences in the gene

What about the second alternative above, copying the nearest sister chromatid? This method fixes the DNA break differently; it requires another DNA as a template containing at least a similar sequence. The DNA break can be repaired by copying the neighboring, matching DNA.

Here is the kicker, we can introduce a DNA molecule that we have produced on the lab bench and fool the cell into thinking that this sequence belongs to the replicated sister chromatid. The DNA sequence we introduce will compete with the sister chromatid to act as a template. So, using this technique, we can manipulate which type of mutations we want to cause in our target sequence.

In this way, we can, for example, study what happens without a certain protein. Or what happens if we insert a designed sequence into the DNA? Or if we produce point mutations that activate a gene? As you will see in future posts, there is a lot we can do with these tools. But for now, let’s quickly look at the most common genome editing tools available.

The most used genome editing tools and how to cut DNA – a speed date with the tools:

Meganucleases – the George Harrison of genome editing tools

Endonucleases that recognize and cut specific DNA targets. They recognize groups of 12-40 nucleotides and are highly specific. Meganucleases exist in nature but can be modified to target your gene of interest.

THE GOOD: Highly specific, which means they can cut where we want to cut and nowhere else; that is low off-target effects.

THE BAD: Meganucleases require modifications to target your specific sequence of interest. The approach can be a bit complicated to implement because of this.

Zinc finger nucleases (ZFNs) – the uncle that used to have it all (but times change)

These endonucleases have been fused to a DNA-binding protein domain called the zinc finger domain. We can make changes to zinc finger domains so that they target our gene of interest, and that is how we direct the DNA-cutting endonuclease to specific locations of the DNA. Each zinc finger domain recognizes and binds to triplets (a sequence of three nucleotides). By linking different zinc finger molecules, we can target specific sequences of triplicates.

THE GOOD: Zinc finger nucleases can be used in several organisms.

THE BAD: Target-specificity can be complicated to achieve. Remember that the Zinc finger domain needs to be made in such a way that it recognizes our piece of DNA.

Transcription activator-like effector nucleases (TALENs) – the one-hit wonder

Similar to ZFNs, TALENs target the endonuclease to a specific DNA target through specific DNA-binding motifs. However, compared to ZFNs, each TALEN DNA-binding motif recognizes only one nucleotide.

THE GOOD: Compared to ZFNs, TALENs are easier to use and can be applied to different organisms.

THE BAD: Still not very easy to produce. Possible off-target effects.

This is where we are now! Everybody is talking about it, including the gym bros.

We explain CRISPR in more detail in another post, but as a summary:

CRISPR is derived from the bacterial immune system. The endonuclease of the CRISPR system forms a complex with a so-called guide RNA. The guide RNA contains a 20-nucleotide sequence that recognizes a chunk of around 20 nucleotides in DNA. The endonuclease cuts the DNA upon binding.

THE GOOD: Easy to design. Cheap. High efficiency. It can be used in the cell-culture dish or in vivo.

THE BAD: May be subjected to off-target effects.

Attention: The figures are schematics and do not represent the real mechanisms behind DNA association and cutting. They are meant to give the idea behind endonucleases and are therefore disproportional and simplified in all ways (endonucleases do, for example, not carry giant scissors on their sides).

So, there you have it. That’s genome editing, and that’s where we’re at. Don’t think for a second that this is the end, though. There are multiple applications to CRISPR and more to come. We will for sure experience loads of criticism, optimism, and misunderstandings. After all, it’s a pretty controversial tool in some instances. No matter what, stay tuned, and the Ivory Embassy will do its best to keep you updated.

Until then, ask questions and enjoy life.