thank you so much for joining me today i'm very excited to give you a sneak peek of a new lab from mini pcr bio it's called knockout and it's a hands-on lab where students actually use crispr cast to modify dna inside living cells before we get going i want to point out that some of my colleagues are monitoring the chat so if you have any questions during the webinar you can ask them there my name is alison and i work on the curriculum team at mini pcr bio and before that i taught high school biology mini pcr makes affordable biotechnology equipment and innovative lab curriculum at mini pcr we strive to create labs that give students hands-on experience with aspects of biology that were previously inaccessible for students in the classroom crispr cass is an excellent example of this everyone has heard of crispr and it was probably my student's favorite topic in biology class but the best we were able to do was watch videos about crispr and use simulations now with our new lab your students can actually use crispr cast to modify dna in living cells to give you a sense of what we'll cover in today's webinar we'll start with some background on genome editing and the crispr caste system we'll spend most of our time talking specifically about the mini pcr knockout lab that lets you use crispr cast genome editing first let's cover the background before we dive into crispr i want to take a step back and put things into context crispr cast is used to perform genome editing which is when scientists change the dna sequence inside a cell this can include adding dna sequences or removing dna sequences scientists can even change specific bases like swapping an a for a g dna contains the instructions for life so changing an organism's dna can change the organism because of this scientists have long sought to edit the genome for example we could correct a disease-causing mutation or even introduce new traits to an organism like drought tolerance and crop plants while scientists have had some success with genome editing in the past the methods were difficult to use and required a high level of expertise genome editing was also quite expensive and very time consuming between 2012 to 2013 work from several scientists came together to demonstrate the use of a powerful new way to edit the genome the crispr caste system compared to other methods of genome editing the crispr cast system is relatively simple and easy to use crispr cast is also adaptable and in theory can be used to target any dna sequence in any organism for genome editing finally the crispr cast system is also specific meaning it allows scientists to precisely control which dna sequences get modified within a cell's genome i cannot overstate the impact of the development of the crispr caste system as a genome editing tool in fact in recognition for their contributions two of the scientists that pioneered the technique were awarded the 2020 nobel prize in chemistry while crisprcast is used by scientists to edit the dna in living cells scientists did not invent the system crispr cast occurs naturally in bacteria and archaea where it helps these microorganisms recognize and destroy invading viruses it was the realization that we could apply the crispr caste system as a tool to edit the genome of any organism that was a game changer in its most basic form using the crispr cast system for genome editing requires two major components first you need a cast protein cast proteins are a type of nuclease enzymes that cut dna like molecular scissors while there are several different cast proteins scientists typically use one called cast 9 for genome editing the thing that makes cas9 different than most other nucleases is that a small rna molecule that binds to cas9 determines which dna sequences cas9 will cut this is the second thing you need the small rna is called the guide rna because it acts like a guide and tells cas9 where to cut the dna one quick note you might be familiar with the fact that while dna tends to be double-stranded rna tends to be single-stranded rna can take on complex secondary structures for example in the guide rna you can see that there is a portion where the same rna base pairs with itself to form a loop rna can also base pair with matching single stranded dna which you'll see soon one last note you might have noted that while we need cas9 and a guide rna i didn't mention any crispr the crisper part of the system is actually specific to the antiviral defense in bacteria and isn't involved when scientists perform genome editing a more accurate name would be cast genome editing but i have to admit that crispr is a lot catchier so let's walk through how the guide rna and cast 9 work together to illustrate the process we'll look at a simplified schematic where the dna is untwisted just because it makes it easier to see what's happening first the guide rna binds to cast 9 cast 9 unzips the dna allowing the guide access if the guide matches the dna cast 9 will cut the result is called a double strand break because both strands of the dna are cut after a double strand break occurs there are a few different things that can happen depending on the system in the organism even in eukaryotic cells that have more advanced ways of repairing double strand breaks the most common thing that happens is that when the dna is repaired random mutations are introduced and the gene is disabled this might sound like a bad thing but it can actually be useful for scientists disabling a gene is a major way that scientists determine the function of a gene this is something we'll talk a lot more about in a minute there are also instances where disabling a gene can be useful to treat a disease we'll come back to an example of this at the end of the presentation it's really important to understand that crispr cast gives scientists a simple way to target a precise dna sequence for modification to understand how the guide rna specifies which dna sequence cast 9 will cut we need to add a little more detail to our diagram at one end of the guide rna is a special sequence of approximately 20 bases only when this 20 base rna sequence finds a complementary dna sequence will cast 9 cut the dna to better see how the guide rna and the dna can be complementary let's zoom in you can see that the yellow guide rna matches the gray dna here following the base pairing rules normally when we think of base plane rules we think of dna where a binds with t and c binds with g but like i mentioned single-stranded dna and single-stranded rna can also bind rna has used instead of ts so when there's an a on the dna the matching rna base will be a u you can see this in the first base pairs here when reading left to right but let's keep the big picture in mind which is when the guide rna finds a complementary dna sequence cast 9 will cut the dna and that the result is that both strands of the dna are broken it's easy for scientists to produce pieces of rna with any sequence of bases so all you need to do for crispr cast genome editing is that this is design a custom rna guide that is complementary to the dna sequence you want to modify then you introduce cas9 and the custom guide rna to cells let's take a look at one way scientists use the crispr cast system many news stories about crispr cast focus on scientists working to edit the genome to correct disease-causing mutations while this is absolutely an important use of crispr cass it is not the most common use of the system there are still many many genes whose function remain unknown a common method to understand the function of a gene is to disable it and then observe the effect scientists call this knocking out a gene if we illustrate the process very schematically we can say we have some gene genex and we want to know what it does if we knock out gene x and observe what happens it's often possible to deduce the function of the gene crispr cast genome editing made it possible for scientists to easily knock out any gene in any organism this is the most prevalent use of the crispr cast system in scientific research and it's led to an explosion of important discoveries across all fields of biology i can't help myself and i'm going to take a minute to tell you about one of my favorite quirky discoveries that came about thanks to scientists using the crispr cast system to knock out a gene in 2016 a snail made a big splash in the news most snail's shells spiral to the right but jeremy the snail had a shell that spiraled to the left because of snail anatomy jeremy would be unable to mate unless another lefty snail was found in this picture we can see a more common righty snail whose shell spirals clockwise or to the right and here is jeremy the famous lefty snail whose shell spirals anti-clockwise or to the left while scientists knew that some snail shells spiraled to the left and even had some hypotheses about which genes controlled shell spiraling there was no way for them to test their ideas this is because for reasons that are too complicated to get into in this webinar scientists did not have a practical way to knock out genes and snails that is of course until crisper cast genome editing which works in all organisms scientists were able to use crispr cast to knock out a gene called lsdia1 that they thought controlled shell spiraling scientists used cass in a custom guide rna designed to match the lsdia1 gene to knock out the gene and snail embryos and the result was lefty snails definitively showing that the lsdia1 gene controls the way that snail shells spiral even though this might seem like a weird thing to get excited about this research is really interesting because before crispr casts there is no practical way to knock out jeans and snails because crisper cast makes it so easy to edit the genome in any organism it's led to an explosion of discoveries like this one for those of you that don't get as excited about understanding snail developmental biology as me which i concede is probably the majority of people i will add that getting a better understanding of the ways that left right asymmetry can develop might have clinical applications in humans for example in a rare condition called cytosine versus the normal left right asymmetry of the human body is mirrored causing the heart to be on the right better understanding the different ways that left right asymmetry can be established even in simple organisms like snails could help us better understand left right asymmetry in humans now that we've walked through an example of scientists using crispr casts to knock out a gene i want to circle back to something we talked about earlier i told you that one of the reasons crisprcast revolutionized genome editing was that it allows scientists to target specific dna sequences this is critical and i want to take time to discuss it in more detail remember that only when the guide rna finds a complementary dna sequence will cas9 cut the dna and that once the dna is cut a common outcome is that the gene will be disabled with all these details it's easy forget to forget that all this is happening inside living cells with the snails scientists added cas9 and the guide rna to very early embryos which developed into snails with the gene knockout when you are modifying the genome it is absolutely essential that whatever method you're using is very specific otherwise you could be introducing unwanted mutations to the genome which could have negative consequences one of the biggest advantages of the crispr cast system is that it's pretty specific that's because for cas9 to cut 20 bases of the of the guide rna need to match the dna while that might not seem like a lot the chance that any random 20 based dna sequence will occur is actually around one in a trillion to put that into context the human genome is only 3.2 billion bases long so most guide rna should only match one location in a genome if any i don't want to give the impression that the crispr cast system is a magic bullet that will solve any problem though while crispr cast has made it easier to modify the genome of living cells even human cells there's still a lot we don't know and the vast majority of scientists and doctors don't think we should be using crispr cast to modify the human genome yet except under some very specific conditions and i'll give one example of this at the end of the webinar before we move on from our snail tangent i'm happy to report that they did find another lefty snail for jeremy to mate with and here is a picture of jeremy with one of his snail babies now that we've covered the background behind crispr cast genome editing let's dive into a preview of our new lab knockout first we'll discuss the framing for the lab then we'll walk through the experiment and the expected results and we'll wrap up with a preview of the free educational resources from mini pcr that are available to complement this lab in terms of framing this lab focuses on the most fundamental use of the crispr caste system to edit the genome so the goal is to disable or knock out a gene in bacteria in terms of understanding how we how we will accomplish this there are two important things to discuss the first is how will we introduce cast 9 to the bacteria while crispr cast does occur naturally in many species of bacteria the e coli bacteria used in this lab do not naturally contain cas9 so we need a way to introduce cas9 bacteria have their own genome but they can also naturally carry small rings of dna called plasmids scientists take advantage of plasmids and use them to deliver dna to bacteria while bacteria can naturally take up plasma dna a process called transformation this is a very rare event luckily scientists have figured out ways to make bacteria more likely to take up plasmid dna a common method is to expose the bacteria to calcium chloride and then briefly heat shock the cells this combination makes the bacteria more likely to take up plasmid dna we will transform the bacteria with a plasmid that contains the cast 9 gene and the instructions for a guide rna and the cells will use this plasmid to produce the cas9 protein and the custom guide if you've never done a bacterial transformation before don't be intimidated it's actually really easy there are some incubation steps so i won't show you the whole procedure live but i will demonstrate the key points to give you a preview of what will happen in the video i think of a bacterial transformation as having four main steps step one is to prepare the bacteria you add the calcium chloride and the plasma dna step two is to heat shock the cells this combination of calcium chloride and brief exposure to heat should make some of the bacteria take up the plasma dna that we added step three is to allow the bacteria to recover from the ordeal of being heat shocked we add some very nutrient-rich media to make the bacteria happy and then we give them a break and step four is to spread the transformation reaction on a solid nutrient agar plate and select for bacteria that were select successfully transformed i will explain the theory behind this in just a minute give me a second to switch to full screen video and then i'll talk you through the highlights of the lab protocol now let's see the transformation protocol in action i'm going to set up two reactions in the tubes with the purple labels one is a control and one is my experimental sample but for simplicity i've labeled my tubes one and two the first thing i'm doing here is adding calcium chloride to my tubes remember that we're using calcium chloride to help get the plasmid dna into the bacteria scientists don't know for sure how calcium chloride makes bacteria more likely to take up plasma dna but it's hypothesized that it makes the cell membrane more permeable and scientists have been using this method for decades next i will add the bacteria which are in the brown glass vial the cells come freeze dried and all you need to do is rehydrate them the day before you want to do the transformation you don't even need to grow the cells in the incubator all you do is add prepared media to the file of freeze-dried bacteria and leave it at room temperature overnight then the cells will be ready for transformation the next day i just added the bacteria to my first tube and i want to warn you that normally scientists add the calcium chloride to the bacteria and then incubate them on ice for a while then they add the dna and heat shock the cells here you will see me add the dna before the incubation step just because it's more convenient to do all the pipetting at the same time and it doesn't make a difference in how well the transformation works so here goes i'm adding the plasma dna remember one sample is a control and the other is my experimental sample and i promise i will explain them shortly i admit that i have a lot of experience with pipetting but setting up these two reactions took me less than two minutes because all you need to do is add three things to the tubes calcium chloride bacteria and the plasma dna so i just added plasmid dna to my second tube and i'm flicking it gently to mix and that's all you need to pipette these tubes will sit on ice for 30 minutes to give the calcium chloride time to work its magic then we will heat shock now i have a low-tech water bath set up you can use a heat block or an actual water bath if you have one but a cup of water and a thermometer get the job done all you need to do is heat track the samples at 42 degrees celsius for 90 seconds and then it's back on ice for two minutes the combination of calcium chloride and the heat shock should allow plasma dna to enter some of the bacterial cells but it's also kind of harsh to help the cells recover we add a very nutrient-rich media to the tubes and allow the cells to grow at room temperature longer incubation times tend to lead to better results and that's actually pretty convenient because you can leave the cells like this for more than 24 hours if that works better for your class schedule i really like protocols that have flexibility like this built in since it can be hard to get a lot of bench work done if you have a lot of students in your class and you only have a 45 minute class period so again i'm just flicking lightly to mix and then i'm going to leave these tubes at room temperature the last step is selection even though we made it more likely that some bacteria will take up the plasma dna it's still a relatively rare event and we need a way to get rid of all the bacteria that were not transformed scientists use antibiotics to do this which at first seems counter-intuitive since antibiotics kill bacteria but the key is that when you transform plasma dna the plasmid carries an antibiotic resistance gene if you spread the bacteria from your transformation reaction on nutrient agar that contains that antibiotic then only the bacteria that took up the plasma dna and carry the antibiotic resistance gene survive and grow on the plate if we spread the cells out diffusely enough and give them time to grow then a single bacterium will divide enough times so that it forms a cluster of genetically identical bacteria that will be visible to the naked eye scientists call these colonies so you can see that i'm about to add my transformation reaction from my second sample to the plate it's actually really easy all you need to do is pipet some of the transformation reaction onto the auger and then you use a plastic spreader to spread it around the key is that you don't need to apply any pressure you just glide the spreader over the surface and i prefer to turn my plate like a lazy susan and keep my spreader in the same position and that's it you turn the plates upside down so that the auger is on top this prevents condensation from dripping down onto the auger if you have an incubator the next day you will be able to see colonies and if you don't have an incubator it just takes a little longer for the colonies to be visible i know i just explained selection while it's plating the bacteria but it's important concept so i'm going to recap it here with some illustrations when we heat shot the bacteria in the presence of calcium chloride it makes it more likely that the bacteria will take up the plasmid dna but there are millions or billions of bacteria in the tube and most of them won't take up the plasmid to get rid of the untransformed bacteria we plate them on nutrient agar that contains an antibiotic remember the plasmid contains an antibiotic resistance gene so only the bacteria that took up the plasmid should survive now that we've discussed how bacterial transformation is used to introduce the components of the crispr cast system into the bacteria let's discuss the gene we are targeting for knockout the goal of the knockout lab is to use the crispr cast system to disable a gene and bacteria because we want the results of gene knockout to be really easy for students to observe we chose to target a gene associated with a clear visual phenotype the black c gene the lac z gene contains the instructions for making a protein called beta galactosidase or beta gal for short beta gal protein breaks down lactose but it can also break down a chemical called excal to produce a blue pigment if you grow bacteria on nutrient agar that contains excal any bacteria with a functional maxi gene will appear blue on the other hand if the lac c gene is disabled then the cell won't be able to produce beta gal protein without beta gal excal will remain colorless and the bacteria will appear white this clear difference makes it easy for students to tell if they've successfully knocked out the black sea gene using crispr cass blue colonies means the lacie gene is functional while white colonies means the lack c gene has been disabled now that we've gone over the background for the experiment and you've seen how it's set up let's walk through the expected results remember i set up two transformations now i'm finally going to talk through the details of the experimental condition and the control condition as we just discussed black sea is a great reporter because of blue white screening in this experiment the waxy reporter gene is in a plasmid that is already present in the bacteria before you start the experiment so the bacteria that you saw me transform already had the lacse reporter gene in the experimental condition you transform the bacteria with a plasmid that contains the extr instructions to make cast 9 and a guide rna that is complementary to the lac c gene once the plasmid is inside the bacteria the cell produces the cas9 protein and the guide rna the guide rna binds to cas9 then the complex searches the searches for dna that matches the guide rna remember that because our goal is for cas9 to cut the lacie gene the guide rna in this experiment is designed to be complementary to the lac c gene cast 9 will cut the lag c gene disabling it the question is how can we tell that this has happened remember we're targeting the lag c gene because we can use blue white screening to tell us if the waxy gene is functional so stop and think to yourself what color do you expect the bacterial colonies to be in this experimental reaction since the lag c gene is not functional the cell cannot make beta gal protein and excal will remain colorless if we grow these bacteria on a nutrient agar with excal we will observe white colonies that tells us the lac c gene is disabled now let's walk through the control in this reaction you transform the bacteria with a plasmid that contains cas9 and a random guide rna once the plasmid is inside the bacteria the cell makes cas9 and the guide rna the guide rna binds to cas9 then the complex searches for dna that matches the guide rna but the guide rna is not complementary to the lacz gene so the lacz gene remains functional and the cell can make beta gal protein then the beta gal protein will process excal and produce a blue pigment if we grow these bacteria on a nutrient agar plate with excal we will observe blue colonies that tell us that the lacci gene is intact to really drive this home let's look at the two reactions side by side when students add cast 9 and a guide rna that is complementary to the wax c gene cast 9 cuts the lac c gene and disables it leading to white bacteria as a control students add cas9 and a random guide rna the guide is not complementary to black c so the lag c gene remains functional leading to blue bacteria now i'll show you how easy these results are for students to interpret in the control with cast 9 and a random guide the black c gene is functional and the bacteria are blue adding cas9 with the lac c guide rna disables the lacie gene leading to white bacteria this lab lets students experience first hand how a custom guide rna and cast 9 are used together to knock out genes in living cells if you want to take this a step further you can do an optional pcr genotyping add-on so your students can confirm using molecular evidence that the lacz gene has been disabled in lane two we're looking at a control reaction which is a positive control for the presence of the lac c gene in lane 3 we're looking at another control reaction which is a positive control for the presence of the cas9 gene in lane 4 we see dna from the control transformation where the bacteria were blue the pcr reveals the presence of both the lac c dna and the cas9 dna which is what we would expect because the guide is random it wouldn't cut the lacz gene in lane 5 we see the results from the experimental reaction where the bacteria were white the pcr reveals that the cas9 dna is present but the lac z dna is absent indicating that it has been cut and disabled to put all of this into context so you can get some sense of how you could implement this in a class the whole procedure including the prep can be done in as little as three days the first day is teacher prep on day two students do the transformation and on day three students can observe their experimental results we really wanted the teacher prep for this lab to be simple and streamlined teachers only have to make one type of nutrient agar plate and the bacteria for this experiment are easy to prepare as i mentioned the bacteria come freeze dried and all you need to do is add liquid media to the cells and let them recover overnight your students will transform the rehydrated bacteria and there's no need to streak starter plates this means you can get all of the prep done in less than an hour and you can do it all on the same day we also wanted the lab to be accessible for high school students in general biology so the student protocol is quick and simple your students complete the two transformations we discussed and grow the bacteria from both reactions on the same type of plate the transformation can be completed in as little as a 45-minute class period and your students can observe the results in as little as 24 hours if you grow the bacteria at 37 degrees celsius we'll wrap up the webinar by highlighting some of the free educational resources from mini pcr that complement this lab first i want to highlight dna dots dna dots are two page explanations of molecular biology topics written using non-technical language mini pcr has a whole library of dna dots available for free including one on crispr cass to help students better grasp the way the guide rna and cas9 work together we've also created two paper activities for students to model how the crispr cast system works one of the most exciting things about crispr cast genome editing is how it's been used to make major breakthroughs in both basic and applied science to dive into a fun example of basic science research we have an activity where students model the research on snail shell spiraling that we discussed earlier in terms of using crispr cast to treat disease you frequently think of the more complex use of crispr casts to introduce a specific change in the dna instead of using the system to knock out a gene but scientists took the more basic knockout approach in the first patient to receive crispr cast gene editing to treat sickle cell disease the paper model lays out all the background for students to be able to understand this cutting edge therapy here's a little video of the paper models in action i'll walk you through the paper model in more detail in a minute because showing the model is going to help me answer one of the questions that was submitted in advance we are not able to get into a lot of background about crispr cast today i really encourage you to check out mini pcr's free webinar to learn more about how the crispr cast system acts like a bacterial immune system and the scientific work that led to its application as the genome editing tool there's even a free student worksheet to accompany the webinar you can find all of the free educational resources today that i talked about at www.minipcr.com crispr you can find the link if you expand the youtube description for this video by clicking the show more now i'll answer a few of the questions that were submitted in advance as a former teacher i'm in awe of what teachers are dealing with this year and this is an excellent question if i were to implement this lab in a hybrid setting where some of my students were in person and others were remote i would probably film myself doing the transformation so the remote students could still see the protocol then i would have all my students work in groups to answer the student questions and predict their experimental results on a collaborative digital worksheet the fact that the results are an easy to observe color change makes this lab great for students who need to observe the results remotely another great question that we got was about guide rna design the short answer is that scientists use algorithms to design guide rnas there are lots of free sites out there where you put in the dna sequence that you want cas9 to target and then it gives you a bunch of potential guide rna sequences it is important to understand that not all guide rnas are created equal it's too complicated to get into detail here but i'll try and give you a broad overview on one hand the algorithms try and predict how effective each guide will be at successfully recruiting cas9 to the target dna on the other hand the algorithms try to predict whether the guide rna might also recruit cas9 to other unintended areas in the genome i know i said that finding any 20-base sequence is statistically unlikely but for reasons that we still don't entirely understand guide rnas sometimes bind with dna sequences that aren't a perfect 20 base match these are referred to as being off target the bottom line is that the algorithms make lots of complex calculations and then output a ranked list of suggested guide rnas there is one other constraint in guide rna design that i will mention here earlier i said that the guide rna binds to cas9 and then cast 9 unzips the dna giving the guide access but i left out one important thing cas9 doesn't unzip all the dna in the genome it recognizes a three base sequence called the pam and only unzips the dna there the pam is included in the paper model so i'm going to switch to my cell phone camera and use the paper model to better explain the pam just give me one second the cas9 pam sequence is from 5 prime to 3 prime n g g where n can be any base the fact that the pam sequence is so common is useful because the guide rnas have to be adjacent to a pam here we are looking at a stretch of the lsdia1 gene from snails and i've marked all the pams in purple you can see there are a lot of them on both strands of the dna there are even instances where pams overlap on the model these are indicated with brackets now i will place this dna onto the cas9 protein to show how all these things work together in the paper model i already attached the guide rna onto the cas9 protein to show that cast 9 needs to find a pam there is this purple pam box on the cas9 protein i'm going to slide the dna under the guide rna this allows me to still see the sequence on the top and bottom strands of the dna to model cas9 recognizing a pam you slide the dna until the purple pam box on the cas9 protein lines up with a pam sequence on the dna then you check to see if the guide rna is complementary to the dna so for example here on my guide rna i have a g and the dna sequence across from it is another g that's not complementary so i'm going to slide my dna down and check the next pam sequence you find a pam that works with the guide rna sequence the model will show you where the cas9 nucleus domains would cut the dna so in this position i have a g across from a c and a across from a t another a across from a t in fact if we check the entire guide rna sequence it's complementary to the dna so this is a match i would just take a pen and mark where these arrows are indicating that's telling me where the cas9 nucleus domains would cut the dna and then i take it out and cut it with scissors in terms of guide design the model also comes with a blank guide rna template that you can use to create your own guide rna this gives students a more concrete understanding of one of the constraints of guide design the fact that it needs to be adjacent to a pam sequence okay that's it be on the lookout for the release of the knockout lab in early 2021 you can sign up to receive an email update when the lab is available for purchase this will also enter you in a drawing for a free knockout lab you can scan the qr code here or check out the link in the youtube video description thank you so much for joining me you