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Komplizierte Wissenschaft in einem passenden Format zum Bier – Der Science sl6
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What is CRISPR/Cas?
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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.
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.
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.
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.
How to Stop the Clock
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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.
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.
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.
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
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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.
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.
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?
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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.
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.
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.
Teaching Yeast to Age like normal People Part III
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In the post “Teaching Yeast to Age Part II” I explained what I tried first to teach my yeast to shorten its Telomers at each cell division. I also told you that it did not work yet. While I have not yet completely given up on Tretrad dissection, I decided to first try another way. It would already be nice to have my fluorescently tagged cells with a knocked out Telomerase but it would be a lot cooler to be able to switch the Telomerase on and off at will.
The way I’m trying to achieve this is called promoter shuffle. As you might know, Proteins (like the subunit of Telomerase I’m interested in) are encoded in the genome by a fixed code where 3 letters (called a codon) in the DNA (bases) are translated into one letter in the protein (amino acid). This code is well known and understood. All codes for proteins start with the same start codon and end with one of three stop codons. However to really produce this protein in the cell there needs to be more. The gene needs to be flanked by regulatory code that influences if and how strongly the encoded protein is produced. Most important for this is the so called promoter region. This is the code directly before the start codon. It is normally between 50 and a few hundred bases long. Their code is a lot less understood than that for the protein. There are strong and weak promoters, promoters that are always active and promoters that are switched off or on on specific stimuli.
Since I want to make the Telomerase switchable I take the genetic code for one of its subunits and exchange some of the code before its start codon against something else. There are a handful of common switchable promoters in yeast that are well described but most of them have some disadvantages. I identified a relatively newly identified one that I hope will be a good choice.
It is the promoter of the gene DDI2 that is normally almost inactive but increases its activity more than 1000-fold if the cells are incubated with the chemical cyanamide. In the concentration it is used here cyanamide has almost no effect on the yeast cells.
Exchanging code in the yeast genome luckily isn’t too complicated. Yeast quite readily incorporates genetic material that you feed it if a stretch at the beginning and end of the inserted code match the code in the genome. This process is called recombination. Recombination is also the process that makes children unique instead of copies of their parents because it randomly mixes the genetic material of your parents that is passed on to your kids.
So I need a stretch of DNA containing the 500 bases before the start codon of DDI2 with 50 bases on both ends from the area before the start codon of the Telomerase subunit. I did a lot of thinking how to build this myself but it would have been a lot of work. Then I thought about the paper where I first read about the DDI2 promoter. In the nature article “Fine-tuning the expression of target genes using a DDI2 promoter gene switch in budding yeast” Professor Yu Fu of the Chinese Academy of Sciences and colleagues characterized the promoter and explained how they integrated it in genomic locations.
So I just wrote an email to Professor Fu asking if I could get some of the material they used. He actually replied within a day and said that he’d be happy to help me. He sent me the code of the promoter without expecting anything in return. That’s a great thing about the science community. Most people are happy to share and are really nice if you show interest in their work.
So all I have to do now is add the code stretches at the ends that determine the target where I want to integrate it. I’m doing this with a PCR which I’ll explain in my next article.
Social Media for Science
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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!
Teaching Yeast to Age Part II
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In my last post I explained how to get a bag of 4 yeast spores of which one hopefully has the two mutations I want in my new yeast strain. Now comes the really tricky part: You have to find this spore.
Before I explain the method you have to get an idea on how small one yeast cell is. The average yeast cell is about 3 to 4 micrometers. The smallest distance between two dots to still see them as two with the naked eye is 0.1 mm which is about 300 times a yeast cell. A human hair is on average between 0.06 an 0.08 mm thick, which is 20 times a yeast cell. If you look at the picture above, depicting yeast cells magnified 1000x, the whole frame is about the thickness of a hair.
OK, so they are really small and not visible with the naked eye but we want to find a single spore (that is again smaller than a normal cell) and identify it. The method we use for that is called micromanipulation. A micromanipulator is basically a microscope with a stage (on which you fix the plate with your cells) that can be moved in tiny increments. And it has a lever with a very thin glas needle fixed to it that through some mechanics can also be moved in a very small scale.
My lab has quite a cool and modern micromipulator that has a joystick to move the stage and a computer driving you to positions on a virtual grid on your plate where you can put single cells.
What you now do is you look through a patch on your plate where you’ve put some of your cells. Once you’ve found a bag with 4 spores (which is called a Tetrad) you move the tiny needle really close to it. A moment before the needle would actually touch the plate a meniscus of water forms between the needle tip and the plate that scoops up the Tetrad and hopefully nothing else. Then you move to an empty position on the grid and, by moving and shaking the needle while it almost touches the surface, you try to deposit one of those 4 spores. Then you go to the next spot and do that again. So in the end you get a plate full of 4 spots in a row where you deposited single spores. You can see how it works in the following video.
After finishing the sorting I put the plate to 30 °C for one or two days to give the cells time to grow. The result looks like this:
As you see not all cells I’ve laid out have grown. That is quite normal because not all spores are viable after the procedure. On the left side was the reservoir, where I’ve put a lot of cells to look for Tetrads. I’ve cut that off with a clean scalpel so the cells do not overgrow the rest of the plate.
In the next step now I have to identify which of these cells are really derived from spores (and are haploid) and which of them are actually just normal diploid cells that accidentally lay around with 3 buddies and looked like Tetrads. To do this you use another nice feature of yeast. Wild yeast can live of a lt of things and can make everything it needs (like amino-acids and bases for DNA) semselves. The labstrains you normally use however have some genes they need for this knocked-out. This means to let them grow you need to add some amino-acids and other things to the medium. Now the trick is, that the genetic traits that I want to breed together here are marked with the genetic code that the laboratory strain needs to make these amio-acids themselves. That means I take this plate and stamp it over to a plate where the medium is missing two special amino-acids, only those cells can grow that have both genetic traits that I need. This is called selection.
The problem here is that the diploid cells I mistook for Tetrads also have both markers and can also grow. Here however the statistic can help. Since I always put all 4 cells that were together in one row and in a Tetrad only one of those 4 can have both of the genetic markers, I now have to check my selection plate for rows where only one of the 4 cells has grown. Another test that I can do afterwards is to see if the cells of such a colony mate with other haploid cells of either mating type. Diploid cells won’t do that.
The results look relatively promising but further analysis showed that this actually hasn’t worked. However this is an important part of science: Things don’t work. After trying this a couple of times I decided to first try another way to teach my yeast cells to age that is a bit more elegant and hopefully works better: I will put a switch on the gene for Telomerase so I can turn it on and off as I want. I’ll explain how this works the next time.
Teaching Yeast to Age Part I
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In my last article I explained the aim of my project, which is making Telomere length of yeast visible. I also explained the first problem for this: Yeast holds its Telomers constant by a tightly controlled use of Telomerase. That means it doesn’t show Telomere attrition as a sign of aging like our somatic cells do.
This is why I have to teach yeast to age like normal people first. The most obvious strategy to do this is to switch off their Telomerase. In molecular biology you normally do this by removing or destroying the gene or in this case one of the genes that code for Telomerase. We call this a knock-out mutation or just knock-out.
One of the big advantages of yeast as a model organism is, that there are libraries of yeast strains available where you can not only find yeast strains with any protein tagged with Green fluorescent protein (as I explained last time) but also strains with any gene knocked-out. An exception are obviously genes that are so vital that knocking them out makes the yeast in-viable. The genes associated with Telomerase are a bit special in this because yeast with one of them knocked-out is viable but only for a certain number of generations (about 400). One generation under ideal circumstances is about 90 minutes for yeast. So after cultivating them for about 4 weeks their first Telomere become critically short and their cell cycle is stopped permanently. They go into a phase of senescence like our somatic cells at that point.
So basically it would be easy to just thaw one of these knock-out strains from our – 80 °C freezers and I’ve got yeast that ages. The problem however is that I also need a mutation from another library. As I explained in the last article I want to use Telomere associated proteins that are fused with Green fluorescent protein as a marker. So I’ve got a few options: I can take a strain with one mutation and do the other mutation myself. I’ll tell you another time how that works.
Or I can bring both mutations into one cell by mating the two strains. Now I have to explain another cool feature of yeast as a model organism. You might have heard that all the genetic information in your cells is kept in two versions. One from your father and one from your mother. This is called diploid. It has some advantages to have this. The most obvious is that if an important gene is broken on one of your chromosomes there is still a second copy of it from your other parent that can fix it. On the other side in our germline cells (eggs and sperm cells) there is only a single copy of each chromosome. This is made by randomly mixing the the chromosomes you’ve got from your parents. This is also why each egg and each sperm is genetically different. This is called haploid. This form is obviously practical for sexual reproduction because it stops us from collecting more and more copies of the same information in our cells. But it is also great for me as a molecular biologist because if I want to switch off a gene I only have to do so once. There is no second copy that could make trouble.
Now comes the great thing about yeast: It can stably live in a haploid and a diploid form. There is no male or female yeast but there is something similar: The mating types Mat a and Mat alpha. Haploid (only one set of chromosomes) yeast can be either Mat a or Mat alpha and can only mate with the other mating type upon which they produce diploid offspring and stay diploid. Diploid yeast is both Mat a and alpha but not at the same time. It normally changes it’s sex every time it produces offspring. Diploid yeast only mates with diploid and not with haploids even it it currently has the opposite sex. To make diploid yeast haploid again they need to sporulate. Spores are a dormant form of yeast that the produce in bad times. In the lab you create bad times for them by first giving them ample sugar and than suddenly putting them on a very strict diet.
So how does this all help me produce a haploid yeast strain with mutations from two different strains? I first cross the two mutant strains of opposing mating type from which I get a diploid strain with one of the mutations on one set of its chromosomes, the other mutation on the other. Then I bring them to sporulate by setting them on a diet. In the process of sporulation one diploid cell produces four genetically different haploid spores by randomly sharing parts of their chromosomes from each of their two parents. These four spores stay together in one bag (called the Ascus) until times get better and they grow out again and mate. Now if I’m lucky one of these 4 spores has both of the mutations I want in it.
Now the big trick is finding it and this is done by sorting single tiny yeast cells. I’ll show you this crazy technique next time.
What are Telomeres?
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To explain what I’m doing I’ll first explain what Telomeres are and why their length is relatively important.
Telomeres are solving a problem that came up when bacteria became eucariots (plants, mushrooms and animals). At this point they stopped storing their genetic information on DNA-rings and instead developed linear chromosomes. This brings some structural advantages for bigger chromosomes but has a huge disadvantage. Each time a cell divides (which most cells do a lot) it has to replicate it’s genome so each daughter can have a copy. This is done by enzymes called DNA-polymerases. The process only works in one direction on the DNA-string and doesn’t start at the beginning.
For a circular genome this is no problem but for a linear genome this means the loss of a few bases (the building blocks of DNA) at each devision. For a while this is not a huge problem if you don’t write important stuff at the beginning (or end) of the chromosome. And that is already the first function of Telomeres. Aside from mechanically stabilizing the chromosome ends, they are a peace of genetic code at the ends of chromosomes that do not contain vital information.
This is already enough for many of our somatic cells (those cells in the body that form most of our body) they only divide a certain number of times until a tissue (like a muscle or your brain) is formed and then just work and do not or only rarely divide.
Only cells that need to divide unlimited or often (germ line or stem cells but also unicellular life) need another trick. This trick is called Telomerase and earned it’s discoverers a Nobel Prize. Telomerase is an enzyme complex that uses an RNA-template to elongate Telomeres and so completely solve the so called End-Replication-Problem.
The fact that most somatic cells are not using this trick and have telomerase switched off has an interesting implication. It makes Telomeres something like a cellular clock of aging. They limit the number of times a cell can divide and rejuvenate a tissue before it goes into a state called senescence. The more cells of an organ are in this state, the less likely the whole organ gets to repair itself and to function correctly.
Another type of cell that needs to replicate a lot is cancer. This is why about 4 out of 5 cancer cells have Telomerase switched on.
The full picture of the connection between Telomeres, aging and cancer are quite complicated and still under discussion but it is quite clear that influencing Telomere length has quite some potential to treat cancer on one side and influence aging on the other.
If you’re interested in a good overview on the current state of knowledge on the connection between aging and Telomeres there’s a quite new review article from Vaiserman et al., 2021.
If you want further information on Telomers and cancer I’d recommend the article of Hiyama et al., 2003 about Telomerase as tumor marker or Graham et al., 2017 about Telomeres and prostrate cancer.
For an overview on Telomeres as a target for drug discovery see the nature article of Bearss et al., 2000.