Genetic Engineering

All right. Welcome to tech nights, we are excited to be able to share our session with you which will be talking about genetic, engineering, a quickly advancing, and hot topic, in science, right now. Uh before we get started we'd like to introduce, ourselves. I'm aiden, i'm a graduate student at the university, of pittsburgh. I grew up in colorado, but i did my undergraduate, in oklahoma, and now i've been here in pittsburgh, for about five years. I work in the nacotra, lab and we study. Self-non-self-recognition. Which is a type of immune, response. And a really cool animal called hydrictenia. Specifically, i study a type of a protein interaction, that allows. The animal to recognize, close relatives, of self. Uh, i use a lot of. Different molecular, biology, and genetic engineering, techniques, in my research, some of which we'll talk about today. But i'm also interested in the application. Of computational, biology and data science. When i'm not doing science, and before the pandemic, i enjoy, playing soccer, and going to dance argentine, tango. And now during the pandemic, i go for more walks, and play piano more. Hi my name is alyssa. I'm a freshman, studying computer, science at carnegie, mellon university. I'm from the chicago, area. I like computer science because it lets me solve all kinds of different problems, and programming, is really fun it can be applied to a lot of other fields, including biology. Outside of school i like reading books playing piano and spending time with my friends. All right. So today we're going to cover some basic, dna and protein information to make sure everybody's, up to speed with what we're talking about in the rest of the talk. But then we'll get into genetic engineering, techniques, both in the traditional, research, or small scale applications, such as bacterial, transformation. And then we'll get into the larger, more complicated, applications. Ideally, our goal is to help you understand, generally, how these techniques, work, and then also get used to start thinking about the applications. And ethical, considerations. Needed for this technology. In the real. World. Yeah so let's start with the basics, about dna. Dna, stands for, deoxyribonucleic. Acid. And it's a molecule that carries genetic information, like you might have learned. It's shaped like a double helix, which is what we call this twisted, ladder shape in the picture. The information, stored, in dna, is a code made up of an alphabet, of four bases.

Adenine, Thymine. Guanine, and cytosine. Which we represent, with the letters, a, t, g and c. All cells have dna. From single celled bacteria, to much larger organisms, like plants and animals. And dna, is really important, because your genes are stored in dna, and this determines, traits, like your hair color your eye color, pretty much everything, else about you. Okay, so before we talk about, how dna, actually affects your traits. We have to talk about proteins, so. The way dna, actually affects your traits is through proteins, before we talk about the exact process. Here's a little background, information, on proteins. Proteins are made up of smaller molecules, called amino, acids, a protein, is actually a long chain of amino, acids like you can see in this diagram. Some proteins, are shorter and some are much much longer, up to over, 10, 000 amino, acids. In cells, proteins, are made by, some organelles, called ribosomes. Which create these long chains of amino, acids by attaching, amino acids one by one. So how do the ribosomes, know which amino acid to attach next. So dna, provides the recipe, for that the genetic, information, which we know is stored in dna, actually gets passed through another molecule, called rna. And it ends up at the ribosomes, to create the. Proteins. Uh so. What does dna, encode, we aren't going to focus on the details, of this process in our session so don't worry if you don't understand, everything here. But here's a general, overview of how exactly genetic, information, gets passed from dna, to rna, to protein. So you can think of the four bases. A, t, g and c like the letters of the dna, alphabet. A sequence of letters is nice but letters don't mean anything on their own, in the english language we group letters together to make words. Similarly, in the dna, language each group of three bases, makes a codon. So each codon. Like the name is the code for a specific, amino acid. Remember, amino acids are the pieces, that we link into a chain to make proteins. But the ribosomes, can't read the dna code directly, because the dna is kept in a different part of the cell. So instead, we use a messenger. Called rna. Which is very similar to dna. We copy the genetic, information, from dna, to messenger. Rna. Or mrna. Through a process, called transcription. Then the mrna, goes to the ribosome. And the ribosome, reads the message, this is called translation. Because the ribosome, is translating, the mrnas. Message to make a protein. Another, type of rna, transfer, rna, which we call trna. Is attached, to the amino, acids. So trna. Helps us actually, match the codon, or the three letter code to the right amino, acid. I won't go into much more detail on this but you should know that dna, acts as a recipe, for making proteins. By providing a sequence, of three letter codes that correspond, with amino acids. Yes so, in this image you can see how it goes. From the dna. To the mrna. To the trna. Yeah and then each trna, will correspond, with an amino acid. So, why is this all important, well, this is how your genes, actually, affect your traits. So proteins, do the real work in the human body, and in all organisms. So proteins, help the cell, move, and grow, digest, substances. Transport, substances. All the other things that, make life function. And these variations. In proteins, cause different traits. In humans this can be traits like eye color height and others that you can't see. Um this is the reason, why you, elephants. Birds flowers, bacteria. And fungi, have so many differences. It's all based on the way genes are expressed. And that all comes down to dna, acting like a recipe, for proteins. Okay, so let's do an activity, to review what we just learned. Go to your worksheet. Um there should be a link. In the, description. And do the section labeled, dna, activity. So right now you can pause the video, take as much time as you need, come back and unpause the video when you're done, so go ahead and hit pause. Okay, so now we're going to go over the answers. Okay. Take a moment to check your answers. You can pause the video here again if you need to. So you should have gotten the message, glow-in-the-dark. The first step was to take each dna, letter, and transform, it into an rna, letter, this is called transcription.

So In transcription. T pairs with a, like you see in the first box there, a pairs with u. Then c pairs with g and g pairs with c. So after that the second step was to take each group of three bases. Which is called a codon. And match it with the correct, amino acid using our chart this is called translation. Notice how the first codon. Aug. Corresponded, to the start codon which signals, the start of the message. The last codon was a stop codon which told us to stop reading the message. We. Ended up linking the words together, to make a secret message. Just like how the ribosome, links the amino acids together to make a protein. Okay, so now that we've covered the basics, of dna we'll start talking about how dna, is used, in the first genetic engineering, topic today. Bacterial, transformation. All right. So, in order to, understand. How bacterial, transformation. Works. I'm actually going to give you a little bit more information, about how. Dna is packaged, into cells. So first dna is packaged, into chromosomes. And bacteria. There are one or two circular, chromosomes. But bacteria can also have smaller pieces of dna. Called plasmids, which also, are circular. Um and here i'll just give you a schematic, usually, the chromosomal, dna, is indicated, as some sort of, kind of mess of uh lines there. And then the plasmid, is uh, i'm not sure if you can see this based on where our. Videos, are, but the plasmid, is a smaller circle, circular, piece of. Dna. And then, other cells. Usually multicellular. Cells. Most anything, except bacteria. Are much, have much more complicated. Chromosomes. They usually, have, um. Linear chromosomes. And they end up in an x shape. They're made up of. Um they are made up of dna, but they're. Packaged, a little differently, so within the cell there is an organelle called the nucleus, and this is where dna gets stored. And again we showed you that general dna, structure, which is um. Just that long line of dna. And what happens, is based on its structure it actually forces, it to. Um. To coil, in on itself. And so it starts coiling and what it coils around are actually called histones. And these histones, form a connection. Or collection. Of. Um. Um. A collection, of bundles that get wound into a tight helical, fiber. And then this helical, fiber also gets wound into itself and that is called a super coil. And then that super coil gets, mashed up all together and that's what makes up the chromosome. Pretty neat huh. So, if we look at what a plasmid, is, we're going to not focus on chromosomes, those are just too big so if we look at what a plasmid, is. Um. Uh it's a small circular, piece of dna, and these usually carry one or two genes. Of, interest, that we have interest in um expressing, and they definitely. Occur naturally, in nature, in fact they were originally. First observed, in. Bacteria. And since plasmids, are smaller this actually makes it very easy for them to be copied, and transferred, between, cells. A way that this can happen in bacteria, is through the process, called. Conjugation. And so for example let's say that we have two bacteria. Um the first one which we will call the donor, has. A plasmid. Which we'll call the plasmid, f. Um, and this allows it to find and connect with other bacteria. That. Through protrusion, that's called a pilus. And, it's able to find, other bacteria that don't have. That. That plasmid, in it. And so what it'll do, is. Once it finds that bacteria, it'll make a connection, and they'll come closer together, so that way. Um. A special enzyme called a dna polymerase. Can, amplify. The plasma dna, and transfer, it to the new. Bacteria, cell, and then you end up with two cells that have, um. Have the plasmid, in them now. And so that's how. Bacteria can share plasmids, between each other. So how can we use plasmids, in science. Now why would something this small be so important to how we do science today. Well the relatively, short length of plasmids. Makes them easy to manipulate, and engineer. Plasmids, are usually somewhere between, five to ten thousand base pairs or five to ten kilobases. And that means around five thousand to ten thousand of those nucleotides. In some, order. That happen. Um. Uh, and again those nucleotides. Are a t g and c. Now i know this sounds like a lot but to give you an idea of how small a plasmid, actually is, a bacterial. Chromosome. Is around, 130. 000 base pairs, or, um, 130. Kilobases. Up to, 14. Million, base pairs or 14 megabases. And so if we compare, that, a bacterial, genome to the one which your cells use every day to keep. You alive and doing everything that you do. The human genome is around 3 billion base pairs. And so. Um again, that just kind of dwarfs, the number, of, uh, what we find in, bacteria, and then even what we see in plasmids. And so, um. To give you another example of kind of the size of these things if we think about a plasmid, as a one-page, essay that you have to write for some of your school work.

A Bacterial, chromosome, would be the equivalent of writing a book. And then, the human genome would. Then in turn be a collection. Of britannica. Encyclopedias. Just volumes, of volumes. Containing, all this information. So when we think about plasmids and when we deal with them they're again relatively, small in the large. Scheme, of. Things. So, since plasmids, are so easily designed. Um. Or since they're so small they can be easily designed. So we can easily add, a gene to a plasmid, which, can then be expressed. We can express things like antibiotic. Resistance. Or. Any genes that we're interested in researching, and of course, genes that express, proteins. So these traits, make plasmids, very useful, and easily transferred, into bacterial, cells and when there are transferred. This process, is called. Transformation. Because the bacteria, becomes, transformed. By the new plasmid. And so in order for transformation. To occur, bacteria, must be, what is called. Competent. And what that means, is that they are able to accept. Foreign dna. Into them. And so bacteria, can be competent, by a couple of different ways. Uh first of all they can be. Competent, naturally, from their own dna. Or. They can have competency. Induced. By either, using, chemical, means, such as calcium, chloride. Or even using electricity. And. Once one of these has occurred. There are holes, or areas in the membrane, around the bacteria. That allow foreign dna to enter. Such as a plasmid. And so once that plasmid, has entered or we mix the bacteria and plasmid, and then once it is entered. We can leave the holes. We can't leave the holes in the cell wall, so they're procedures, to help the bacteria, close up those holes and then voila, the bacteria, has been transformed. Once its membrane, is uh. Intact, again. But, transformation. Is never a hundred percent, efficient. Um so if we were to, transform. A bunch of bacteria. Of competent, bacteria with and put it with a bunch of plasmid. Some bacteria, will get it and some would not. Now if we culture these bacteria. To get them, to multiply. What would end up happening. Um, or, what, the bacteria would end up doing, is we'd have some with and without. But the ones without the plasmid, would actually end up multiplying. More. Um and this is because. The plasmid. The bacteria with the plasmid doesn't have, the plasma doesn't, contain, anything that it needs in order to survive so there will be less bacteria, with the plasmid. Again it takes a lot of resources, in order for the bacteria, to replicate, dna. So it won't drag the plasmid along if it doesn't need to. Instead what we can do to make sure that our plasmid, with the plasmid, grows is we can introduce. Antibiotics. Into the mix. And so in order to do that we don't want to kill all the bacteria, so what we do, is in our plasmid. We encode an antibiotic. Resistance. Gene. And so. Um. So when we do that and then we, apply. Um. Apply an antibiotic, to the growth medium that these bacteria, grow in. Uh what will happen, is that. The bacteria. Will be exposed to the antibiotic. The one without the plasmid, the bacteria, will be able to kill and it will not be able to reproduce. Or multiply. Um. But the plasmid. Or the the bacteria, with the plasmid. When the when the antibiotic tries to destroy, it. The plasmid. Expresses. The antibiotic, resistance, gene that we gave it, and that enzyme, is able to either, destroy, or bind to the antibiotic, trying to kill our bacteria. And, the the antibiotic, will then be neutralized, so then we'll get, more and more of. Our bacteria, with the plasmid. Okay, so now we're going to try bacterial, transformation. Our project, is going to be to make bacteria. Glow, under ultraviolet. Light. So here's some materials, that we'll need for the experiment. The first one, is. Lb, agar, so that's luria, broth, agar, it's a gel-like, substance, we're going to put in our petri dishes for the bacteria, to grow on, and the lb, contains, some nutrients, that bacteria, need to survive grow and reproduce. So that's how we culture, bacteria. So then um.

Ampicillin. Which we're going to abbreviate, as amp is an antibiotic. Like aden was discussing, on the last slide that means it kills bacteria. Doctors, sometimes, use antibiotics. Like ampicillin, to treat patients who have bacterial. Infections. So it gets rid of bacteria. But we're going to be using, it to eliminate, the bacteria, that we don't want in our petri. Dishes. Our last ingredient. Here is arabinose. Which we're gonna abbreviate, as ara. It's a sugar, so. Um, it can be used for energy. In our experiment, we're going to use arabinos, to trigger a certain gene to turn on. Okay, so the name of the plasmid, we're going to transfer into our bacteria. Is p-glow. And we're going to use the bacterial, transformation. Process, that aiden, explained in the last few slides. So our plasmid, has three genes. The first gene, is called amp r. It makes bacteria. Resistant, to ampicillin. Which is our antibiotic. Remember. That means that bacteria, that have this p-glow, plasmid, won't be killed by ampicillin. So the second gene gfp. Is the green fluorescent, protein, gene so when this gene is turned on it causes bacteria, to glow, or fluoresce, under ultraviolet, light. So how do we turn gfp, on, the answer is that some genes actually, control. Other genes. So the third gene, rsc. Regulates, the green fluorescent, protein, gene, so when the arabinose. Sugar is present. This gene will turn on the green fluorescent, protein gene. Okay. A final note before we do our experiment, here when we say plus p glow that means p glow is present. That means we perform, bacterial, transformation. On the plus, picobacteria. When we say, minus, pico, that just means we didn't genetically, modify these bacteria, so p glow is absent. So. The minus. Bacteria, don't have the p glow plasmid, the plus public bacteria, might have the plasmid. Okay, so our experiment, will have four plates. Or four petri dishes, it's going to be your job to figure out which bacteria. On which plates are going to survive. And also which ones are going to glow, under ultraviolet, light. So, on plate 1 we have lb auger remember that's our nutrient, broth or our food for the bacteria, culture. Plate 1 is labeled minus, pico so we did not perform bacterial, transformation, on these bacteria, so they don't have the plasmid. So on plate 2 we also have lb, but we've added ampicillin. Remember, that's our antibiotic. Plate, 2 is also labeled minus p-glo, so these bacteria, don't have the plasmid. On plate 3, we have lb. And ampicillin. And it's labeled, plus p-glow, so that means we transform, these bacteria. Some of them will have picked up the plasmid, you can see that little yellow circle. But not all of them, so we have a mix on this plate of bacteria, with p-glo, and bacteria, without p-glow. On plate number four we have lb, ampicillin. And we've also added arabinose.

Remember, Arabinose, is the sugar that triggers, the arrow-c, gene on the plasmid, to turn on the green fluorescent, protein, gene. Um this plate is also labeled plus p glow so some of these bacteria. Also have the plasmid. Okay, now take a moment to go to your worksheet. Uh pause the video and try to answer the questions, and we will check the results of our experiment, after you're. Done. Okay. So let's take a look at our results. Okay. Interesting. So on plate one, none of the bacteria, had the plasmid, and there was no antibiotic. So not much happened here the bacteria, survived, because there was no antibiotic, to hurt them, and they didn't glow because there was no plasmin, to give them the fluorescence, gene. Okay on plate 2 all of the bacteria, were killed. This is because we added ampicillin. But none of the bacteria, had resistance, to ampicillin. Because we didn't give them the plasmid, with the antibiotic. Resistance, gene. On plate, 3 some of the bacteria, were killed and some survived. The bacteria, that managed to pick up the plasmid, were able to survive, because they were resistant, to ampicillin. But the bacteria, that didn't pick up the plasmid, were killed by the ampicillin. So that means the only bacteria. Left on the plate are bacteria, with the p-glow, plasmid. So, this, allows us to see why ampicillin. Is useful, so including ampicillin. Allows us to select, for the bacteria, we want. So that we make sure all the bacteria. In our culture have the gene we want, the p-glow, plasmid. But notice that these bacteria. Are not glowing. Because we did not turn on the green fluoresce, protein gene it's just sitting there it's not making any fluorescence, proteins. Okay so look at plate four something very interesting happened. Just like plate three adding ampicillin. Allowed us to eliminate, bacteria, without the plasmid, so the bacteria, that didn't have the plasmid were killed. But adding, arabinose. Our sugar allowed the bacteria, to glow, remember, that when this sugar is present the green fluorescent, protein gene on the plasmid, gets turned on. So only the bacteria. On this plate, the ones that had the plasmid. And were in the presence, of arabinose, this sugar, were able to glow under ultraviolet, light. So if you tried that bonus, question on the worksheet which asks why we need to bother including, plates that don't have the plasmid, the reason is because our experiment, needs a control. So even though only the bacteria. In plate four glowed, we needed plates one two and three for comparison. So without the other plates we wouldn't know that not all bacteria, glow under uv light. And if we didn't have plate 3 we wouldn't know for sure that the gene only turns on when you have arabinose. So experimental. Controls, are very important, to a well-designed, experiment. Okay so this is what the piglow, experiment, looks like in real life. Notice how plate 1 contains, bacteria, but plate 2 doesn't because we eliminated, them with ampicillin. Plate 3 contains, some bacteria, but some have been killed, by ampicillin, and none of them are glowing. Plate 4 is the only one where the bacteria. Are lighting up. Okay. That's it for the piglow experiment, and bacterial, transformation. Now we're going to talk about genetic, engineering, more generally. All right. So now we're going to talk about some larger applications, of genetic, engineering. Genetic, engineering, is defined, as the direct manipulation. Of an organism's, genes using biotechnology. As technology, has progressed we've been able to get more specific, and more accurate with how genes are edited, and i'm going to show you two ways. How it has been done, in science. The first tool we have as scientists. To edit dna, is through. Naturally occurring proteins, called, restriction, enzymes. These are thought. Or there many different kinds of restriction, enzymes, that exist in nature. Many have been isolated, from bacteria. But all restriction, enzymes, recognize, a very specific. Dna, sequence. These sites are called restriction, sites. And every restriction, site has a sequence, made of what's called a palindrome. And the enzyme, can digest, or cut the dna. Only at that specific, sequence. A palindrome. Is a word or sequence, that reads the same, backward, as forward. So for example. The word, level, contains, the same order. Of letters when you read it backward, and forward. The word nurses, run, also reads the same. Backward and forward and you can also make a sequence, of numbers, in a palindrome, such as one two three three two one. Now, one type of restriction, enzyme. Uh digests, or cut dna. Which results, in blunt, ends of the dna, and what i mean by that.

Is When the enzyme, cuts the dna the two sides of the ladder are cut at the same point. One example of this is the, alu1. Enzyme. Which recognizes. The four base pair sequence. Agct. And again, this is the same forward. Um, on the one strand as it is on, going the other way on the other strand. It cuts right between the two middle bases, and you end up with these, equal, length, sides, of the ladder. Now these, ends can be reconnected, with each other, using enzymes, to repair the break. But since that's a blunt end. It can each end can also be reconnected. To another blunt end, of dna which may not may or may not have the same sequence, and so. Sequences, can be recombined, this way. There's another type of enzyme, which results, in sticky, ends of dna. And what these enzymes, do is cut the dna ladder at equal points but on opposite, sides, of, the, palindrome. For example. This enzyme, called. Bam, h1. Or i call it sometimes bam high for short. It recognizes, the six base pair sequence. G-a-a-t-t-c. And when this enzyme cuts the dna, it does so right after the first guanine, on each strand. And this makes, uneven, ends. Uneven, ends in the dna. Now this dna can only be reconnected. With other sticky ended dna. That has the same exact, sequence. So it's much more limited with what it can be recombined, with. Now as cool as restriction, enzymes, are there are a few drawbacks, to using them. First of all a restriction, enzyme will cut sites, with, all sites with that sequence, you can't just pick and choose which site it will cut. So in order to use these properly, you have to know the entire sequence you're digesting, to make sure, that you are getting. The expected, fragments, of dna that you want. And as the length of, a dna, sequence, increases, the likelihood, there will be multiple restriction, sites also increases. So for example, in the human genome which remembers, around 3 billion base pairs, there could be thousands, of places, where, a single restriction, enzyme, could cut. Thus restriction, enzymes are not very useful for genome, editing, as they have too many off-target, effects. Now restriction, enzymes, are, very useful, for plasmid, engineering. So for example, if we look at that plasmid, we used before, in our plea, on our plea glow experiment. For example if we had placed, two restriction, sites before and after. The gfp, or green fluorescent, protein. We could get a p, a piece of dna with from a different gene, such as a red fluorescent, protein, or rfp.

That Has the same restriction, sites on both ends. And then if you digest, the dna, you could make and, recombine, the sequences, you could make a new plasmid. That expresses, the red fluorescence. Red, rather than the, green. But science doesn't really stop there you know this is actually a very limited application, we want to be able to target things specifically. And so, when we want to or how we want to be able to. Engineer, dna specifically, and efficiently. Is by using, um, you know improved. Uh, improved, techniques. And one of these techniques. Um that which has really been developed in the last decade. There have been a lot of. Advancements. So you may have seen some of these things in the news. But the epitome, of gene editing, or genetic engineering. Right now, is a technique, referred to as crispr. And crispr stands, for clustered. Regularly. Interspaced. Short, palindromic. Repeats. Now we heard palindrome, before so we kind of know what that is it's um the same sequence forward and backward. Um. And, uh clustered, means there's multiple, together. Uh regularly. Interspaced. Means that there's a regular, distance between them. And of course these are very short palindromes. So. Sometimes, when you hear about crispr you may hear crisper, cast 9. Or crisper cass. Cast just stands for crispr, associated. Protein. Now the crispr system was identified, in bacteria. Originally. And it was actually identified, as part of. Its immune, system. With, uh, again based on finding all the dna repeats, in the genome. So if we look at a timeline, and how, crispr. Has, um. Essentially, you know taken over science a little bit if we look at the number of publications, with crispr in the title. Back in 2000. Which is probably, before, some of you were born. Which makes me feel really old, but, back in 2002. The crispr, acronym. Was first introduced, into the literature. Then in 2005. Crispr was associated, with the bacterial. Immune defense. Now in 2012. Um, gene editing, applications. Were proposed, for other organisms. And then from there it really skyrocketed. Throughout science, and throughout. Many different organisms. And now we have several thousand publications. A year with crispr being used. In fact uh my lab published, a paper in 2018. With work we were doing with crispr so we are one of the 4, 262. Papers that year. But now probably the coolest, thing, uh or, uh yeah one of the coolest things i can show you about this timeline. Is that here in 2020. Jennifer doudna, and emmanuel. Charpentier. Uh two of the lead investors investor. Investigators. For crispr, have been named the nobel prize winners, in chemistry, which is super exciting. This is the first time in history, of, uh science, nobel prizes. In which. Two female scientists, have been awarded, the prize. So i know this may seem, bleak and overwhelming, with everything going on right now but this is really exciting, news and you should be, um. It should, you know. Broaden your mind and open doors for where women can go in science. Um. So anyways, getting back more to crispr. Again the reason we want to use crispr, the purpose behind, it, is um, that it's genetic, and engineering, allows. For a very specific, very, unique, gene editing. We don't have to rely on restriction, enzyme sites we can essentially, customize, or tailor. Where we want the crispr, sites to go, or where we want our, edits to go. And so crispr, allows us to mutate, genes.

To Study many different things. Crispr, allows us to customize, where genetic engineering, takes place and gives us more options, on what we can engineer. For example, beyond. Just. Studying, what a particular, gene, does. We can create bacteria. Which are very cheap and easy to grow. To produce, insulin. And this can then be used, for. Patients, with diabetes. There are some tools which have been genetically, modified, which we'll talk about in a few slides, but there are also a lot of interesting, applications. For. Personalized, medicine. Which is kind of a hot topic in the science medical field right now. Of course there, are. Applications, for genetically, modified, organisms, we mostly hear about these with food and plants. Um. But basically. Uh, you may have seen a lot of things, in the literature, about, how crystal will. Revolutionize. Everything. And some of these claims are, true or they do have some possibility. But. Claims like such as uh. You know no hunger no pollution, no disease, is a pretty, lofty, goal for any one particular. Technique, and while these goals are good to have. Not one thing even crispr, will be able to is a fix it all solution. All right now let's talk about how crispr, works. First. Scientists, create a genetic sequence, called a guide rna. That matches, the piece of dna, that they want to modify. Second, the sequence, is added to a cell, along with a protein, called cas9. Which acts like a pair of scissors, that cut dna. Third, the guy rna, will uh, homes in on the target dna sequence. And cas9, cuts it out. And then once their job is complete, the guide rna. And cas9, leave the scene. And then finally another piece of dna, is swapped. Into the first place and the old dna, and enzyme, repairs, the cuts and then voila, you've got edited, dna. Now this is not as easy as everyone makes it out to be this is obviously an oversimplification. For example my lab had a very difficult time getting it to work appropriate, in our model. But um, plus there are times when it can have off targeted, effects. And so. Meaning that it doesn't, just edit the site you want it to. There's many reasons, why this could happen but the most important, thing, is that any piece of dna, that has been edited needs to be validated. And any off a target, effects, accounted. For. All right now we're gonna move, um. Now that we know a little bit about crispr, how crispr works we're gonna do some application, based thinking. So before, beginning this section please read the following, or listen as i read it out loud. Um, first. First disclaimer. Any technology, regardless, of its use or discipline. Should have boundaries, in place to ensure it's safe and ethical, use. These boundaries, may need to be revised, and updated over time based on the expanding, knowledge of the technology, for example. Um. If you think about asbestos. Or radium, initially they were unregulated. And ended up causing a lot of problems, and then regulations, were put in place to make sure that they don't cause future, issues. A second disclaimer.

The Points, uh the points of view in the following slides are no way exhaustive, and only represent, a small portion, of a much larger discussion. Uh the information, presented, in the following slides does not necessarily, represent, the values. Uh or, nor is endorsed, by, tech nights. Now the goals, in looking at ethical, considerations. Or the purpose of the following slides, is to simulate, discussion, and critical thinking. In no way. Uh, is the material, shown. Meant to sway. Anyone's, views, religious, moral dietary. Whatever you want to think about, and is only meant to serve as an opportunity. For, discussion. Um. It is not uncommon for everyone to have a different viewpoint, on something, and not everyone has to agree and that's okay. It's important to acknowledge, that every viewpoint, has pros and cons. Um, and then the ethical, use of technology, especially, genetic. Engineering. Uh, engineering, or editing, is currently an undergoing. Discussion, in the science community, today. And we are glad that it is an open conversation. All right with those out of the way. Um. For the next five to ten minutes, uh go ahead and pause the video and think about the next two questions. First what benefits, could, genetic, engineering, have. And then second how could genetic engineering, be harmful. As we mentioned before it's important to identify, both the benefits, and harms of any technology. You can think about these in many different ways see what you can come up with. And again go ahead and pause the video and we'll go over some ideas when you come. Back. All right i hope you have some thoughts jotted down or ideas about these two things. Again there are lots of possible, answers and ideas we're just gonna cover a few common ones that you may have heard. When talking with your family or friends. First genetic, engineering, can be used to improve, plant nutrition, and. Insect, or pesticide, resistance. This could save a far save a farmer's crops from being devoured by insects and allow, us to be able to eat every day right. Genetic, engineering, could treat or cure, certain genetic, diseases, such as tay sachs disease. If you don't know what tay sachs disease is. It's, caused by a single base pair mutation. Um. And what this mutation, ends up causing, is that the the brain, can't metabolize. Fatty acids. And then this ends up resulting, in a person's, death, um so again that one single base pair if only we could change it. It would fix a lot of things. Uh the single base pair. Or. Sorry. Another possibility. Is that organisms. Such, as. Bacteria. And fungi can be engineered. Um. Or they can be used in different medical therapies. Uh or they can be engineered to improve, recycling. Um their different recycling, capabilities, i'm sure you guys have heard. About. Bacteria. Being able to. Eat. Or digest, certain types of plastic. Now just as important as the benefits, let's look at some potentially, harmful, outcomes. First, genetic, engineering, could contribute to the rise of superbugs. Immunodepression. Or toxicity. In humans as well as animals. Second a genetically, modified, organism, could give, an it an unfair, advantage. Over an indigenous, organism. Uh which would result, in, the genetically, modified, organism. From potentially, becoming an invasive, species. Um. And out competing, the indigenous, one. Uh lastly, one thought to keep in mind is, that once something, is genetically, modified, and out in the real world. Uh, rather than in a controlled, lab setting there's actually really no way to get it back it'll always be out in nature, somewhere. Um so that's just one thing to think about, with introducing, genetic engineering, into the world. Now if you look around you could find. News articles, talking about all of these things. My advice is to be careful, of everything that you read online, as you all know, there's a lot of misinformation. Out there so don't trust, everything that you read, but i think it is important to know what's out there and understand the pros and cons. And if you read enough from all points of view you can make more well informed decisions, about, um, your own views and how these things are progressing. In uh society, today. All right, we're gonna do a little case study. This will be another discussion, and thinking question. Or questions. So, we're going to talk about genetically, modified, crops. If you don't know much about them here's a little bit of background. They were first introduced, in the 1990s. Which is.

20 To 30 years ago now. The main genetic modification, introduced, was insect resistance, and herbicide, tolerance. Essentially meaning. Insects don't want to eat it and you can spray weak, weed killer, without it killing your crop. Um these were primarily, applied to corn. Soybeans, and cotton, which are the three. Largest crops. And importantly. These are all federally, regulated. So, joe schmo on his farm in the middle of nowhere, isn't coming up with some random modification. That can get into. Um, the rest of the world. Um. Which is uh again important. It's not just um. You know, random it's a it's a very well regulated, system. Um. So there. So. We're going to come up with some ideas for these next two questions. First of all what are some of the benefits, of using gmos. And second what are some of the potentially, harmful, outcomes, of using gmos. So go ahead and pause the video, and we'll go over some ideas when you come. Back. Okay, so let's go over a few, answers that we came up with, again your answers may be different and that's great we're glad that you're taking the time to think about this and discuss, it, um, first what are some benefits, of using, gmos. Uh one thing that has been claimed, is that gmos, can increase. The overall yield of produce. I read a couple articles, on this and it's not like a three-fold. Increase. Or anything, it's, a pretty marginal, improvement you know maybe about a four percent increase which you know could be important. Um. But, uh it's interesting, an interesting benefit nonetheless. Uh drought resistance, is another, interesting, improvement, that could be made. Any kind of tolerance, to less than ideal weather, would be good especially. So for example, heat tolerance or soil, nutrient, tolerance. Another application, which has been in the news recently, is proving, the amount of carbon, or is. Is improving, the amount of carbon. Which plants can sequester, which is related to the increasing, worry, about. Climate change because of too much carbon in the atmosphere. Now what are some of the potentially. Harmful outcomes of using gmos. We mentioned this before but it could increase the number of superbugs, for which we don't have any. Treatments. In relation, to increasing, crop yields and herbicide, resistance. Most gmos, are commodity, crops which means that they aren't really solving, food shortages, in the world. In fact because of their surplus, they're prodding. More unneeded, items on the market. So for example in the us a lot of high fructose corn syrup is used as a sweetener or sugar substitute, but most other countries, ban the use of it because it's, of, its potentially adverse health effects. By creating pesticide, resistant, plants, this may also support. The production, of unnecessary. Or wasteful, things that we wouldn't need otherwise.

So That's just, some ideas that we came up with, i'm sure you guys came up with other things we'd be interested in hearing about them. Um but we hope, that you've enjoyed our session, we have reached the end of our genetic engineering, talk. Uh we hope that you learned a little bit about how and genetic, engineering, is done in research, and how it's being applied, in the real world through medicine and food. And don't take our word for it, do a little of your own research and dig up something new today. Be sure, to think about pros and cons with anything you look at as well as their. Applications. There's always more information. Out there if you want to learn more so you can go to these links or google it. Yourself.

2020-11-12 01:43

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