Gene editing | How to cut DNA and manipulate genes
Before jumping into the CRISPR technology, we need to learn how researchers cut DNA through different 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 now 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 is easy to use, takes little time to learn and it is cost effective. Most importantly it can alter genes and gene expression of cells in the culture dish as well as 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 wish to increase sport performances.
If the concept of CRISPR is brand new to you, no need to worry, we’ve got you covered in this post. But before storming into the CRISPR field it’s best if you 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.
⊕ 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 cells’ own 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 as well as 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 is part of genome engineering the opposite is not necessarily true.
The DNA damage repair
Before going into how the genome engineering or genome editing works it is wise to understand a bit about the DNA repair mechanisms that are 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 be able to tackle these challenges, the cells have developed a repair mechanism that can repair these breaks. We call this the DNA damage repair. Improperly repaired DNA breaks can lead to 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 basically gives up.
Apoptosis – Programmed cell death. The cell decides that it’s not worth it anymore and starts signalling for pulling 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 better understand the outcomes of DNA damage repair. Imagine a double-stranded break, caused by irradiation.
Several responses are possible:
1) The double strand break is “glued” together properly and it looks like 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 which can lead 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 DNA damage related topics. For now, with this brief introduction, we think that you are ready to understand genome editing and eventually CRISPR.
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 own repair machinery to do what it’s intended to do, which is fixing 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 are able to direct these nucleases to specific locations in the DNA by linking them to some sort of DNA-binding module. The type of module used dependent on the genome editing tool that we use when we want 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 sequence of the DNA. How we do that depends on the tool that we are going to 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 that we want 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 in the lab bench and basically fool the cell to think that this sequence belongs to the replicated sister chromatid. The DNA sequence that 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 in the absence of a certain protein. Or what happens if we insert a designed sequence into the DNA? Or if we produce point mutations that activates a gene? As you will see in future posts, there is a lot we can do with these tools. But for now, let’s have a quick 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 recognise and cut specific DNA targets. They recognise 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 that 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 are endonucleases that have been fused to a DNA-binding protein domain called 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 recognises 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 recognises 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 the use of specific DNA-binding motifs. However, compared to ZFNs, each TALEN DNA-binding motif recognises only one nucleotide.
THE GOOD: Compared to ZFNs, TALENs are easier to use and can be applied on different organisms.
THE BAD: Still not very easy to produce. Possible off-target effects.
Clustered, regularly interspaced, short palindromic repeats (CRISPR) – the new, hot $#*!
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 recognises 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 of it 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.