Category: SciComm

What is CRISPR/Cas?

What is CRISPR/Cas? why is it so revolutionary and can it be used by anyone to easily alter the genome of anything? About a great tool in genetic engineering, what it can and cannot do.

When the Nobel Prize in Chemistry went to Jennifer Doudna and Emmanuelle Charpentier in 2020, nobody was really surprised. When I saw Emmanuelle Charpentier at an award ceremony in 2016, it was clear to most that the Nobel Committee was just waiting for an agreement in a patent dispute to nominate the two scientists. The work that has made them known worldwide is the article “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity” which appeared in the journal Science in 2012. It describes the use of a bacterial defense system, now known as CRISPR/Cas, in genetic engineering.

Emmanuelle Charpentier, one of the developers of CRISPR/Cas, at the Gairdner Award ceremony in the Canadian Embassy in Berlin on September 6th, 2016, own picture

What is CRISPR/Cas

But what is CRISPR/Cas and why is it such a giant leap in genetic engineering? CRISPR describes certain repetitive motifs found in the genomes of a wide variety of bacteria. In the early 2000s, it was discovered that these motifs are related to a defense mechanism in these bacteria that protects them from viral attacks. If a virus infects a cell, it injects its genetic material and thus reprograms the cell for its own purposes. The cell now produces new viruses according to this blueprint and in most cases destroys itself. Viruses are a huge threat to bacteria, which do not have specialized immune cells like we do. So they developed many strategies to counter it.

One of the most widespread of these defense systems is CRISPR/Cas. Certain CRISPR-associated (Cas) proteins bind the DNA introduced by a virus, cut out small pieces of it and insert them between repeat sequences in the bacterium’s genome (CRISPR). If the bacterium is attacked again by this or a similar virus, another Cas protein can now immediately recognize the viral DNA with the help of these stored recognition sequences and cut it before it can multiply.

Revolution in genetic engineering

The two Nobel Prize winners have deciphered this part of the adaptive immune response of the bacteria: The programmable cutting of DNA sequences. And they developed a method to change this programming at will. The trick is that the Cas protein cuts DNA whose sequence matches an RNA sequence to which it is attached. This so-called guide RNA can be produced relatively easily and thus the exact target at which the DNA is to be cut can be set.

Guide RNA and CRISPR-associated protein Cas9 (white) from Staphylococcus aureus, CC von Thomas Splettstoesser (www.scistyle.com)

Since this target programming is very precise, these molecular scissors (in contrast to the tools that were previously available for genetic engineering) can cut the genetic material in a living cell at exactly one point. Today other similarly precise gene scissors have been developed but they are not nearly as easy to program.

Cuts or breaks in DNA often occur naturally. So there are repair mechanisms in each cell to close them. The simplest way to rejoin the two ends of a cut DNA strand is non-homologous end joining. This links two DNA ends without any regard to errors or wether they fit or not. Unfortunately this does usually lead to a few individual letters (base pairs) lost from the DNA sequence or a few new, random ones added.

Small error in the code, big effect – the knock-out

If this sequence is a gene, i.e. the blueprint for a protein, this is devastating for this blueprint in two out of three cases. The reason for this lies in the language of DNA, the code. Every three letters (bases) in DNA form a word. The words are amino acids, i.e. the basic building blocks of proteins, which in turn are the whole sentence that is in a gene.

The problem with writing this way is that there are no spaces that determine where one word ends and the next begins. As a result, there are three possible places to start reading, which lead to completely different words and phrases. A special three-letter word, the start codon, defines the start of reading, i.e. translating the DNA into a protein. The end is again determined by a special word, one of three possible stop codons.

This type of code means that if a single letter or two is added or omitted the entire meaning of the rest of the sentence changes because the reading frame shifts. As a result the protein encoded by the gene is no longer produced correctly. So it will be turned off.

In genetic engineering this is called a knock-out.

Adding new code – the knock-in

If you don’t just want to destroy a gene, but want to insert new information, you take advantage of the cell’s other repair method: Homology directed repair. This is actually the better way for the cell to repair breaks in the DNA, as it is less prone to errors. If the cell has a piece of DNA available that has the same (or similar) sequence as the pieces before and after the cut, the cell incorporates that piece and swaps out the broken piece.

This method can be used to insert a new sequence into the genome of a cell. I have explained how to do this here.

With both methods, the sequence originally recognized and cut by the Cas protein is removed and the genome is not cut again after the repair.

Applications from medicine to agriculture

So what can we do with CRISPR/Cas? Since it made targeted changes in the genome of a wide variety of organisms significantly easier, the progress in genetic engineering was vastly accelerated: In research, the function of a gene can be examined quickly by switching it off. In agriculture, plants can be made more resistant to drought and pests by incorporating resistances from other plants. The first genetically modified animals are also being developed, such as a catfish that is supposed to become more resistant to diseases with the help of a gene from the alligator.

In medicine, the first gene therapies will probably heal regular patients in 2023. The first therapies are mostly carried out outside the body. For example, blood stem cells are isolated from patients with congenital sickle cell anemia, a defective gene is repaired and these stem cells are then injected back into the patient. These can then make functioning red blood cells in the patient, which they were previously unable to do.

Principle of CAR-T-cell therapy, CC by NIH

Another important group of gene therapies that work in a similar way are CAR-T-cells. These are the patient’s own immune cells, which have been taught by (by introducing the gene for a special receptor) to recognize and destroy cancer cells more effectively. Several thousand CRISPR/Cas-based gene therapies are currently in the clinical trials.

So CRISPR/Cas is just beginning to change many areas of our lives.

Can everyone change genes with CRISPR/Cas?

CRISPR/Cas is often described as an extremely simple tool that anyone can use in their kitchen to alter the genes of humans and other organisms. Of course it’s not that simple. On the one hand, the “simple” of a molecular biologist is often anything but simple and in addition to a lot of knowledge still requires special equipment and chemicals that cannot be found in a normal kitchen.

Second, it’s one thing to change a unicellular organism or germ cell of a mouse. However, it is quite another to specifically change a group of cells or even all cells in a finished multicellular organism such as a human being. The only tissues in the human body that we can currently access reasonably effectively with gene therapies are the liver, bone marrow, and the eye.

To illustrate that it is not that simple, this diagram shows what the workflow for a single genetic modification in the very simple unicellular baker’s yeast Saccharomyces cerevisiae looks like. The system and also various images of the Ellis Lab are used here.

Schematic workflow of a CRISPR/Cas experiment in yeast, modified from Ellis Lab

How to Stop the Clock

grayscale photo of man thinking in front of analog wall clock
Photo by Brett Sayles on Pexels.com

Can we slow or even reverse ageing?

The good news is, slowing aging and gaining more than ten years is absolutely possible and doesn’t cost you anything except some discipline (yeah, I know, that’s the bad news here). However, to keep you reading, I’ll tell you a little later how that works. Whether it is possible to slow or even reverse aging is currently the topic of a lot of research.

But let’s start at the beginning. If you want to treat something, you first need to define it clearly. The most obvious definition for aging is chronological. However, changing the actual flow of time falls more into the realm of physics and is probably not very practical. What we want to influence is the biological age. To measure this, we use a lot of different methods, called “clocks,” and they work with blood parameters, heart rate variability, epigenetics, simple photos of your face, or other data. Clocks are all somewhat linked to chronological age but can generally tell you how one (or several) aspects of your biological age compare to the average person your age. So they tell you if you’re younger or older than you actually are.

The Hallmarks of Aging CC from Rebelo-Marques 2018

What is aging?

The specific unpleasant cellular effects of aging are summed up as the 9 Hallmarks of Aging that you see above. I won’t go into detail, but they are all interconnected and lead to what we recognize as aging, like wrinkles, grey hair, loss of muscle mass, frailty, dementia, decreasing bone density, and all the other stuff that you’re not keen on having.

Reading this list, you might already guess why treating aging might have other perks than just living longer. The biggest deal, not only for the individual but also for society, would be increasing the so-called healthspan. It can be argued that while in the last 100 years we have already more than doubled the average life span, the healthspan, the time lived in good health, hasn’t grown accordingly. Our current medicine has become very good at treating most of the countless ailments that old age brings; however, many are more managed than cured. So wouldn’t it be better (and cheaper, by the way) to treat the underlying cause of most illnesses instead of each of them at a time? The results of healthspan research could  revolutionize medicine and bring us from fixing what’s broken to preventing the breaking.

Lifestyle Interventions to Slow Aging

But how far along are we? Will we still get old like our grandparents? That depends. To cite one of the leading minds in this field of science, Professor David Sinclair: “It’s easy to expand your lifespan. […] If you do the right things, which is: Don’t overeat, eat less often during the day, do some exercise, don’t smoke, don’t drink! That alone gives you, compared to people who don’t do that, 14 extra years. So living longer isn’t hard, it just takes some discipline.” Well, I told you, it’s not too easy, but it’s doable.

Especially the eating less often part seems to be important. Intermittent fasting (best more than 16 hours without food) gives the cells a feeling of food scarcity and switches on certain survival programs. Probably the most important is autophagy which let’s cells recycle accumulated proteins and other reserves. This kind of a cleaning helps get rid of things that can cause trouble when they accumulate too much.

The other main effect is a reduction in metabolism and especially on cell division. Since cell division is on multiple levels the main reason for mutation (errors in the DNA) it is also the main reason for aging. Avoiding strong mutagens like smoking, excessive drinking (one drink a day seems to be positive) and sun bathing is helpful for the same reason.

Enough sleep and some exercise have also been shown to positively affect aging in human studies.

However, there is obviously more to aging research than the typical advice on living a more healthy lifestyle.

Different times for intervention to delay illness and death by delaying aging, Figure CC from Douglas R Seals, 2014

Supplements and Drugs

First of all, there are drugs and supplements  that (at least in animal models) show a huge potential to give another few healthy years like Nicotinamide Mononucleotide (NMN), α-Ketoglutarate (AKG), Resveratrol, Metformin, and Rapamycin. I won’t go into detail on those now, but I’ll write some more articles about that on my blog soon.

Most of these, however, seem to work mainly as a prevention and not a cure. And while they show a lot of promise in animal models, so far reliable data from humans is scarce. Most of them work through mimicking food scarcity which can also be reached through fasting.

Senolytics

But there are other measures in the pipeline. An interesting idea is the so-called “Senolytics.” Instead of killing themselves as damaged cells normally do, some become senescent. Senescence occurs when cells sense an instability of their chromosomes after having divided a certain number of times or because of high stress (due to their Telomers), so they permanently stop dividing. Senescent cells also secrete signals that lead to inflammation, changing the development of their surrounding cells and the extracellular matrix.

The more senescent cells in an organ, the less vital and functional the organ becomes. Senolytics like Dasatinib and Quercetin are substances that target and remove these senescent cells to rejuvenate the organ. There are ongoing clinical studies on human patients with these substances on several age-related diseases, and they show some promise, but there is still a lot of research to do.

Cellular Reprogramming

The idea that sounds probably most impossible but has the potential to slow the clock and actually reverse aging is cellular reprogramming. Each cell in our body has basically the same genetic information, the same construction plans packed into our DNA organized in chromosomes. But how does a cell in your brain know that it’s not in your foot and has to behave differently? And, even more important, how does a cell know that it’s not supposed to copy itself as often as possible or try to build a new complete clone of you? The answer is epigenetics (mostly). Epigenetics is quite a young field that has made huge progress in the last 15 years. Epigenetics determines which of the genes of a cell’s genome are switched on and switched off by modifying the DNA or proteins associated with the DNA. These bookmarks make a cell behave as it does. They are changed by environmental influences like sunshine, smoking, food, no food, or a thousand other things. Most of these factors and time itself lead to an overall decrease in these bookmarks, although certain areas of the genome also acquire more of them with time. So the idea is to reset these bookmarks to a “younger” state.​

Yamanaka Factors

In 2006 a set of four transcription factors (regulators for genes) were identified that can reset a differentiated cell from being part of a certain tissue to a very similar state to that of the cells you find in an embryo. The cells treated with the transcription factors become stem cells and can be reprogrammed into almost any cell type within the body. These transcription factors are called Yamanaka Factors after one of the authors of this study from 2006. Using the Yamanaka Factors, there have been successful reprogramming studies on animals. The aim is to reset the epigenetics of cells to young without dedifferentiating the cells, making the tissues they form fall apart. This technique is currently tested to restore vision in primates after successful tests on mice that have gone blind because of glaucoma. David Sinclair’s group carrying out these experiments expects it to be ready for human clinical trials within this year. If this is successful, it would be a new hope for many blind people and be a proof of concept for rejuvenating a tissue by epigenetic reprogramming.

This is however a very ambitious time line and I dare say it won’t happen. The main reason is that the Yamanaka factors used here are some of the most potent oncogenes. Those are genes responsible for the transformation of a cell into a cancer cell. It is to be expected that therapies working on a thin line between dedifferentiation and cancer will be looked upon with extreme scrutiny by the authorities before being accepted for human trials.

Making and using induced stem-cells from a patients biopsy to heal genetic or other deceases or even target aging, Illustration CC by Manal Hadenfeld, 2020

A possible future application of this could be to treat a patient’s cells outside the body to become stem cells and then inject them to regenerate damaged tissue or to rejuvenate the patient as a whole.

Much is unclear about reversing aging. Many studies in the field show contradicting results, but what would have seemed impossible 20 years ago is rapidly evolving from promising basic research to clinical trials. Currently, you still need some discipline and changes to your lifestyle if you want to increase your lifespan and healthspan. However, the more life and health you win through your life choices, the closer scientists might be to real solutions to all the unpleasant effects of aging and maybe to aging itself.

This post has first been published as a guest post on the blog BoldedScience.com and has since been modified and updated.

Transforming Yeast or how to make a GMO

Actual footage of me changing the genes of a hapless yeast, own work

In the last article I ended with a DNA fragment, that I created with a PCR. Now I need to get this DNA into my yeast cells to teach them to age like normal people. This is called transformation. There are several different transformation techniques but most common are elctroporation and chemical transformation. You can spark emotional debates if you ask what is better but the more common and generally a bit easier is the chemical transformation. That is also the method I chose for my aging yeast.

How transformation works mechanistically is not completely understood but the main idea is that you have to overcome the barriers protecting cells from their surroundings. These barriers are the cell wall (for all except animal cells who don’t have one) and the cell membrane. The cell wall’s main responsibility is mechanical stability. You can imagine it like a steel frame that prevents the membrane, which you can imagine like a water bomb, from swelling too much until it bursts. On a molecular level it is a relatively wide net, so it’s not a physical barrier for the DNA molecules. What’s problematic however is that it is electrically charged. DNA is negatively charged and would be repelled from the mostly also negatively charged molecules in the cell wall. This obstacle is overcome by adding positively charged ions that neutralize the charges of the cell wall and allow the DNA to get close to the cell’s membrane.

Schematic structure of a budding yeast cell with a special focus on membrane and cell wall on the right, Giulia Coradello, 2021

But than it has another problem which is the cell membrane. The membrane is a double layer of lipids, fat molecules that have a polar and an uncharged tail. This structure is something like a 2-dimensional liquid. It is relatively elastic and allows uncharged molecules that are not too big to path quite well. Since DNA is huge and strongly charged it normally won’t pass easily. To pass this barrier you have to torture your yeast a bit (*laughs manically in evil scientist). You submit them to a sudden increase of temperature to 42 °C from room temperature. Doesn’t sound too bad but yeast likes it best between 20 and 30 °C above 40 °C starts killing them slowly. This is thought to built differences in pressure between the hot outside and cool inside of the cell which is equalized by the formation of pores in the membrane. Through these pores the DNA can now enter the cell.

To avoid another line of defense of the cells which is enzymes that eat DNA, you also add some carrier DNA, which will be eaten first and so protects the DNA you want to get into the cells. As carrier DNA you normally use Salmon Sperm that you boil and then add to the mix. So if you hear molecular biologists talk about cooking some sperm it’s probably not as weird as it might sound.

After overcoming the defenses you now have a lot of dead cells, many that have taken up nothing or only the carrier DNA but you hopefully also have some that have the DNA that you wanted to introduce into them. What happens with this DNA depends on its form. If it is a closed circle and includes some special signals in the code it will be just kept in the cell and will be copied to the daughter cells when the cell replicates. This is called a plasmid and is good if you just want to introduce genes more transiently without permanently changing the genome. If the DNA (like in my case) is a linear open molecule produced by PCR and has ends that resemble a sequence in the genome of the yeast, it can go into the nucleus of the cell, where the genome is kept. There it can jump in while the cell replicates and integrate permanently into the genome.

Now you have to filter for those cells where this has worked from the rest. You do this with selection. You seed your cells to a medium where only those cells can grow that have taken up your DNA. This is done by adding a marker to the DNA which is a gene that either makes your cells resistant to something that is poisonous to the others or that let’s them live of a food source that the others can’t use. I used cells that normally can’t make Uracil (which is a part of DNA) themselves so they can’t grow on medium where there is no Uracil. They only can grow on this medium if they have taken up my construct which includes a gene that helps them make their own Uracil.

The problem is that this integration is very rare. You start with a huge amount of cells, transform them seed them all to the selection plate and sometimes get nothing, sometimes one or two colonies but sometimes, if you’re lucky, you also get something like 100.

Selection plates of the different transformed strains and a control that was not transformed, own work

To verify that the construct is really integrated where I hoped it would, I now need another PCR. I chose primers that normally produce a short fragment of DNA from the region where I want to integrate my construct. If it is successfully integrated the fragment gets longer which I can see in a gel electrophoresis. And that is what I’m currently doing. I’ll soon give you an update when I’ve finally found my aging yeast.

What’s a PCR and how does it work?

In the last article I ended having a DNA template thanks to Prof. Yu Fu and I had a DNA sequence of the region where I want to integrate my DNA construct. So to now I need to copy a piece of the DNA plasmid (ring) that I have and add a stretch of DNA sequence (~ 50 bases) that are complementary to to the point in the yeast genome where I want to integrate that peace. I can do both in one step with a technique called Polymerase Chain Reaction or PCR.

A PCR is a method to produce DNA without using living cells to do it. It is something like a molecular copy machine. The main ingredients for a PCR are an enzyme called DNA-polymerase, which does the copying, dNTPs, which are the building blocks of DNA, the template DNA from which I want to copy and primers. Primers are small DNA peaces (normally about 20-25 bases long) that define the DNA stretch I want to copy. The forward primer (the start signal) is complementary to the sequence where I want to start the copy and the reverse primer is identical to the sequence where I want to stop the copy.

Schematic drawing of a complete PCR cycle
Schematic mechanism of PCR. CC BY-SA 4.0

The picture above is from the Wikipedia article on PCR that also explains it really well in detail. As you see the first step of a PCR is making the DNA accessible for the copying by heating it until the typical double-strand structure splits up into two single strands. For this you heat it to something around 95 °C. Then you cool it down to the annealing temperature which depends on your primers but is normally a bit above 50 °C. To start the DNA-polymerase you now heat it again to something around 72 °C. This sounds like a very high temperature for an enzyme and it is. The trick is that the polymerase comes from the bacterium Thermophilus Aquaticus (or others) that lives around hydrothermal vents that spew water, smoke, acid and other stuff at temperatures of more than 100 °C. So their enzymes are optimized to work in extremely hot and unpleasant conditions.

Now you repeat these three steps over and over again and with each repeat the number of your DNA constructs doubles. This means you have an exponential growth from 1 to 2 to 4 to 8… to a bit more than a million after 20 cycles to a bit more than a billion after 30 cycles. So you create a lot of identical DNA in quite a short time (normally 1 to 4 hours).

The machine that adjusts the temperature in a programmed manner is called a Thermocycler and looks quite unspectacular.

A typical laboratory Thermocycler made by Eppendorf, own work

This explains how I copy the stretch of DNA from the plasmid but I also wanted to add some sequence to the ends. That’s actually quite easy. I just add the sequence to the primers because they are the beginning and the end of my newly created DNA constructs. Now you might ask: And how do you get these primers? Well like most things today. I order them online. There are companies specialized in producing custom DNA. Basically I could also save myself the whole work and just order my whole construct online but that is still quite expensive. While my exceptionally long primers (primer + sequence are about 60 bases long) cost something between 20 and 50 $, my whole 2500 bases long construct would cost about 1.000 $ to order. That is still quite a difference but it gets cheaper and in a few years biologists will probably order a lot more DNA than they create using molecular techniques like PCR and cloning.

Now that I have my PCR product I want to verify if it is the right one. The easiest way to do that is to put it on a gel that lets short DNA pass quicker than long DNA. Than you add voltage and the negatively charged DNA wanders towards the positive electrode and splits up by size. If you add a marker that includes DNA pieces of known size and a compound that makes DNA visible under UV-light (ethidium bromide) you can see what size the fragments that you produced have. That normally gives you a good idea if the experiment ran as expected.

I did all of that and on the first try the result was no visible DNA at all. Than I changed a few parameters and tried again and saw a very thin band where I expected it and a few others where they shouldn’t be. So I changed some more parameters and on the third try I got this wonderful picture of a gel with a strong band at exactly the position where I expected it.

This made me quite happy but now I had to do something with it. I wanted to put this DNA into a yeast cell and make it integrate into the genome. This is called transformation and is quite tricky as well. I’ll explain that the next time.

Social Media for Science

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Photo by Pixabay on Pexels.com

It’s been about two weeks since I started actively using this blog and as you’ve probably seen I’ve been busy. While I started this blog, I also started to think about how to get people to read it. The most obvious answer is social media. Social media channels are made to engage people and show them what you find interesting. For a long time I’ve only been using Facebook for private and LinkedIn for work purposes. I never really used Twitter because I always thought it’s mostly a platform where politics and media exchange views and act like they are the public.

Than I had a seminar about science communication with Susanne Geu. She told me Twitter is an excellent platform for science communication so when I started the blog, I also started to become active on twitter.

After about two weeks of active work on this blog and different social media channels I made the following discoveries

When it’s about clicks on your blog forget Twitter. You can very quickly find a great bubble of people working in a similar area and engage them quickly (from 1 to 70 followers in one week) but people on Twitter like it short. From thounsands of impressions you get a few hundred interactions and from those only a handful are clicks on your links. However it is still a great tool to get in touch and to communicate punchlines.

LinkedIn works about as good as I expected. Since I have a lot of contacts there I get a lot of quite some likes and quite some clicks but no active discussion or interaction.

I originally wanted to keep Facebook private but I still posted my Blog so friends could see what I’m doing and that obviously brought some encouragement. To tell friends, family and people you know what you’re doing you can also use the WhatsApp status.

What works really well to get clicks but also really good discussions is groups in Facebook. Find some good science groups and post and discuss there. This is not too easy because most groups in Facebook are either full of spam or completely inactive but there are some.

I’ll write an update on my experience with social media in a few months maybe then my impression has changed but so far I’d say all these channels have merits and a different target group.

What I did’t try yet because I do not really thinks that’s my audience is Instagram and TikTok. But maybe I’m wrong there so if you have a different opinion on these networks please let me know.

If you want to see what I do on these platforms please follow me on LinkedIn or Twitter!

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