May-2021-SynBio

SynBio FSP May Seminar Series


[Image appears of Owain Edwards talking on the main screen and participants can be seen listening in the participant bar at the top of the screen]

Owain Edwards: And welcome to the SynBio FSP seminar series for today. Before we begin I just want to remind everybody that the session will be recorded and the recording will be made available on the SynBio FSP website. And before we get started I’d like to acknowledge traditional owners of the lands from which we’re presenting today. So, I’m on, I’m in Perth, so I’d like to acknowledge the traditional owners of the Perth area which is the Whadkjuk Noongar people. And there are two speakers, for the Sunshine Coast the Kabi Kabi people and the Jinibara people, and for Geelong the Wathaurong people, as the traditional owners of the land that we’re meeting on today and pay my respect to their Elders, past and present.

And so for those of you who don’t know me, my name is Owain Edwards. I’m the Domain Leader for, in the SynBio FSP, for Environment and Bio-control and I’m really pleased to be offering for you today two of our, two exciting research projects and project leaders that were in the FSP. Both projects have actually finished so we’re going to hear summaries of the projects themselves. 

[Image continues to show Owain talking on the main screen and participants can be seen listening in the participant bar at the top of the screen]

The first speaker today is Nina Pollock from the University of Sunshine Coast where she is now a research scientist. And Nina held a Future Science Fellowship with the SynBio FSP focused on smart biosensing and remediation technologies after completing her PhD in Austria. Her research explored the expansion of synthetic biology into the field of tissue engineering. And that’s what she’s going to be talking about today, in particular novel and multi cellular structures. And so, currently Nina is a research scientist at the University of the Sunshine Coast development rapid diagnostics for the detection of viruses. 

Our second speaker today is Caitlin Cooper. I’m sure most of you know Caitlin. She has a background in genome engineering in agricultural species and after her PhD she joined the Animal Health Labs in CSIRO to do a post doc which was focussed on decreasing the spread of pathogens in poultry. And in particular she developed a novel genome engineering technique called STAGE or Sperm Transfection Assisted Gene Editing. And that was the basis for her SynBio FSP post doc, which was focussed on transferring that technology to, in particular to cane toads and that’s what she’s, one of the things she’s going to be talking about today. She’s also going to be talking about something associated with a new role research scientist within CSIRO which is a new project that’s APaIR funded at executive level looking at functionality of different class enzymes. 

[Image changes to show Owain talking in the top right corner and a new slide appears on the main screen showing CSIRO, USC, UQ and genecology logos and text: From Biological Computation to Tissue-Engineered Pseudo-Organisms for Bioremediation, Dr Nina Pollak, Postdoctoral Research Fellow University of the Sunshine Coast/AFHEA, 11th May 2021]

And so we’ll start off then by, I’ll switch over to Nina and she can begin her presentation which as you can see is titled “From Biological Computation to Tissue-Engineered Pseudo-Organisms for Bioremediation”.

[Image continues to show the same slide on the main screen and Nina Pollak can be seen inset in the top right talking to the camera]

Dr Nina Pollak: Thank you Owain. So, today I kind of want to give you a summary of what I have been up to in the last three and a half years during the FSP Fellowship. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a model of a complex aptazyme on the right and text appears on the left: Complexed aptazymes]

So, I want to start with biological computation which was actually a project led by one of Joanne McDonald, who’s my mentor here at the University of the Sunshine Coast, PhD students, Bradley Harding. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a model of DNAzymes, and a computer generated image of a person playing tic-tac-toe and text appears: Computing with DNAzymes, Self-folding single-stranded DNA molecules with enzymatic ability, which can be controlled, Interactive Tic-Tac-Toe, Discrimination of viral particles]

So, it’s about complexed aptazymes and I will explain to you what that actually is. So, if we talk about DNA computing we are referring to DNAzymes in our case. And DNAzymes are self-folding single-stranded DNA molecules and they have enzymatic activity. And this enzymatic activity can actually be controlled. So, it’s really cool, one of the first things that was done with this DNAzymes is, was done actually by Joanne McDonald, and she played an interactive Tic-Tac-Toe game and as she usually says it was the world’s most expensive, and slowest Tic-Tac-Toe game, and the DNAzymes, so the computer in those case always wins because it started in the middle. And they also made it on the cover of Scientific American with the interactive Tic-Tac-Toe game. So, these DNAzymes were used in a wide variety of things. 

[Image shows two my diagrams appearing of a fluorescence M and a fluorescent E inside a rectangle]

So, another one is in diagnostics. So, for discrimination of viral particles. So, what you can see here is a M for Marburg Virus, and an E that is just played if Ebola was present. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a model of an input molecule at the bottom left and text appears: DNAzyme structure & complexing aptazyme, Key structural domains, Enzymatic core, Substrate binding region – 5’, Substrate binding region – 3’]

So, we wanted to do something else with these DNAzymes. We wanted to actually quantitate a toxin. So, let me first tell you a tiny bit more about DNAzymes. So, you have key structural domains, the enzymatic core – which you can see down here – with some substrate binding region of the five prime site and three prime site. To read the activity of these DNAzymes we use an input molecule and that’s labelled with a fluorescent molecule and a quencher. 

[Image shows an arrow appearing and pointing from the input molecule to a cut input molecule]

So, as soon as the DNAzyme binds, and that’s because it’s complementary here with the substrate binding regions to the input, it cuts it and we get a fluorescence signal. So, that’s the most simple DNAzyme that you can have.

[Image shows a model of a theophylline aptamer, a substrate and a Not logic compliant E6 DNAzyme on the right of the screen and Bradley’s photo can be seen in the bottom right of the screen]

So, what we have done, we have made the system a tiny bit more complicated to help us out, to quantitate theophylline. So, theophylline is our toxin here. So we aim to reduce theophylline aptamer. So if you’re not familiar with aptamers, aptamers are bind… are secondary structures in DNA or in RNA that can bind for example a small molecule. It’s a bit similar to an antibody and an antigen and how they bind. So, we have this theophylline aptamer. Then we have our E6 DNAzyme. So, E6 is just the enzymatic core. It’s named as different types. And now you can see in green this loop here, that’s the receptive loop that can actually recognise this theophylline aptamer. 

[Image shows two more models appearing on the right of the slide showing the results if there is no theophylline present and then if theophylline is present]
So, in case there’s no theophylline present what happens, we have our theophylline aptamer, and we have our DNAzyme and this receptive loop is actually complementary to theophylline. So, it binds it and this binding causes then a disruption of the secondary structure of the DNAzyme. So, now the DNAzyme is basically impaired, it can’t work, it can’t do its job, can’t cut our input, and so we don’t see fluorescence. In case theophylline is present, so a toxin here, it is bound by this theophylline aptamer. It just forms a structure here in this little cartoon. That’s not the real structure it forms. And our DNAzyme is, yeah the structure is as it should be, and it can actually cut the fluorescence part off, the input off, and it’s not quenched any more.

So, as I mentioned this work was done by Bradley Harding, who recently graduated, and we also finally could have his graduation in person. 

So, that’s why I’m using this particular picture of him. Let’s have a closer look at how this really works.

[Image continues to show Nina inset in the top right talking and a new slide appears showing a bar graph on the left and on the right showing fluorescence levels with and without theophylline and a model to detect theophylline in the middle and text appears: In vitro Detection of Theophylline, Three DNAzymes were successfully inhibited by the aptamer, with the aptamer being successfully removed in the presence of theophylline]

So, we wanted to detect theophylline. Use it as a bio-sensor, so first thing as I already told you if the RNA aptamer is present it will disrupt the function of the DNAzyme and we do not get high fluorescence levels. In case theophylline is present we get far higher fluorescence levels, so that’s what’s displayed here in green. So, we have made three different variants and all of them successfully were inhibited by this aptamer in the presence of theophylline. So, what’s important now for biosensor, it’s always good to know if it’s specific right. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a bar graph on the right showing theophylline caffeine and theobromine levels and text appears: Specificity of Ligand, Testing beyond clinically relevant levels, 200 uM, Danger zone for theophylline and caffeine, Lethal dose of theobromine for dogs, Caffeine and theobromine are both indistinguishable from background activity]

So, I should probably tell you a tiny bit about theophylline. In itself, it is a last resort drug that is used in emergency rooms mostly in the United States if you have a severe asthmatic attack. And it is only used there because it is quite dangerous. If you are over a certain level, it’s highly neurotoxic, and if you are under a certain level it’s just completely ineffective. So, you need to be in the perfect range. So, that’s why it’s important to be able to quantitate how much theophylline is in the patient’s blood. So, there are a few ligands that are very similar to this theophylline component, like caffeine, and theobromine, components in coffee and tea. And that’s probably something that lots of us are consuming on a daily basis. So, if we accidentally detect that that wouldn’t be great. 

So, we had to show that our detection system is specific. And as you can see caffeine is not detected and theobromine is not detected, and neither of those three variants. That’s great and further we can also quantitate it. So we can see which levels of theophylline are present in the blood. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a photo of tissue-engineered pseudo-organisms in petri dishes and text appears: Tissue-engineered pseudo-organisms]

However, I’m not going into too much detail, so the next part I want to talk about is the tissue-engineered pseudo-organisms. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing seven photos of real and artificially engineered jellyfish and a stingray and then a video inset of a Free-swimming Medusoid and text appears: Tissue-engineered pseudo-organisms, Artificially-engineered multicellular systems performing organism-like functions, such as movement and sensing]

So, you are probably by now familiar with what I understand by tissue-engineered, pseudo-organisms. So, for me these are artificially engineered multicellular systems and they must perform organism-like functions. And with that I mean something like movement or sensing. So, there are a few very famous examples and one of them is a jellyfish like pseudo-organism. So, it looks a tiny bit like a jellyfish. On the top here you see a real Medusoid, so the younger juvenile form of this jellyfish, and that’s what Parker and his colleagues made. It’s made out of scaffold with heart cells that were isolated from rats and basically layered on top. And you need the heart cells because they contract as our heart beats,  the cells contract, and that’s actually resulting in some movement which you can see here in this video.

So, this jellyfish like pseudo-organism swims in its solution. They also did a fancier version, a stingray, and that is actually even able to follow lights through a maze. And one of the latest examples would be this little, tiny, roundish kind of thing. It’s made out of aggregated frog blastula cells and it can move by itself and in the lower version of this picture here you see these blue tracks, those are actually the movement tracks. And the cool thing here is it’s all made out of cells. And they used artificial intelligence to actually build those little pseudo organisms.

[Image continues to show Nina inset in the top right talking and a new slide appears showing a jellyfish pseudo-organism model and text appears: Key features – Jellyfish-like pseudo-organism, Scaffold material, Biocompatible, Degradable (not PDMS), High degree of batch-to-batch consistency, Cellular system, Unlimited number of cells, Human embryonic stem cells, (not isolated rat cardiomyocytes), differentiated to cardiomyocytes, Structural design, Mimic juvenile jellyfish, Organism like functions, Bioremediation, Self-locomotion]

So, back when I started the project it was the jellyfish that was available, and so I kind of wanted to make jellyfish like pseudo-organism 2.0. So, we wanted to change a few things like the scaffold material for example should have been biocompatible and degradable, excuse me. Yeah and the material needed to be consistent from batch to batch. Then we had the cellular system, the cellular system, we wanted to make that a bit better, and we didn’t want to use the cells that were isolated from animals. So, we sort of, excuse me, so we wanted to use embryonic STEM cells and we used these cells and differentiated them to cardiomyocytes, so heart cells.

They’re… worth mentioning a bit here is that we have no reproductive organs. So, that is really good from a point of social issues and license to operate stuff. We still wanted to mimic this jellyfish. It’s just simple enough but we wanted to give it a function. So, in our case the function was bio-remediation. So, take a toxin and break it down. And we wanted to still move by itself. So, self-locomote. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a diagram of the enzyme detoxification process on the right and text appears on the left: Sensor component miRNA switch]

So, first about the toxin, we need to break it down and we need to sensor it first. So, I will talk about how we achieved sensing and that’s done with the micro RNAs. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing two models with switch targeting and text appears: Ligand-responsive miRNA switch targeting, No ligand – CYPP1A2 expression low, Ligand – CYP1A2 expression high]

So, this is by the way all published now. We have a microRNA switch that we use to actually target this toxin, theophylline. That was our proof of concept component. And the enzyme that we used to break it down was a Cytochrome P450, it was 1A2. So, here you can see a cell with a nucleus. And in this nucleus you have the genetic information for our enzyme and it’s followed by a micro RNA in its unprocessed version. So, primary miRNA. And the important bit here is that we have a structural switching aptamer at the bottom. So, it’s a modular design. It’s the primary micro RNA and it has this aptamer embedded. In case if theophylline is present it actually binds in exactly that region where this aptamer is and because it’s a structure switching aptamer, as soon as the compound is bound it will actually change the structure of this primary micro RNA. And this is here illustrated by it bending to the side. And that means it can’t be processed anymore. So, all this further downstream processing by like cutting it, cleaving it with drosha, and further processing with dicer and so on doesn’t work. And so this primary micro RNA is never incorporated in the RNA using silencing complex and so we don’t see silencing of our gene, so like a whole enzyme. So, we have lots of the enzyme.

And we measured that with luminescence. So, as soon as the component is present we get lots of luminescence, we get lots of the enzyme and so we are ready to degrade and break down the component. So, that’s what you want. So, we achieved, the idea here is to achieve biological computation and by controlling molecular behaviour in vivo, and micro RNAs were just excellent and easily engineerable tools. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing two diagrams explaining the  moderate control and improved control of MiRNA switches and text appears: Key facts: Sensor component, MiRNA switches for toxin-induced control of an enzyme scavenger, Moderate control following an established protocol, Improved control with evolutionary designed version, new design is simple to implement using web-based databases and prediction tools]

So, now a few key facts about this so easily engineerable tools. There was a protocol out there and we just followed the protocol, and we got moderate control over our enzyme and we thought like “OK, how can we make this better? Is there another way? Why aren’t we actually using micro RNAs that were produced by nature and then just plunking in our structure switching elements?”. So, we did that. We used a micro RNA that was meant to target our enzyme and just plunked it in and actually by doing this we achieved improved control. So, in general all these micro RNAs are available and you can find them on the base, databases and you just use some prediction tools that are all free like MiRBase, MIRWalk and the mFold webserver.

[Image continues to show Nina inset in the top right talking and a new slide appears showing two graphs showing the results of improved synthetic control of enzyme production and text appears: Improved synthetic control of enzyme production, based on miR-39a, resdesigned natural occurring miR-378A]

So, to convince you now what I meant, so on the left hand side that is following a protocol that was published and on the right hand side is our process of just taking a naturally evolved micro RNA and re-engineering it. So, I’ll guide you slowly through. Up here on the top you see the micro RNA and how it looks based on this protocol. You have perfect match with this micro RNA. Micro RNAs usually don’t have a perfect match to their gene that they’re targeting. So, you usually have an imperfect match which is the case in the natural occurring micro RNA that we redesigned. 

So, we’re measuring the luminescence. On the x-axis you have increasing levels of theophylline and on the y-axis we are seeing the CYP1A2 activity, in percent that we are measuring with luminescence. So, we have in green the control up here, that’s just the enzyme alone. So, that should be always on, so it’s set to 100%. Then we have the negative control. We shouldn’t see anything that’s like way down here. And then we have our micro RNAs. So, we have a micro RNA that’s a control. It doesn’t have this structure switching at the [19.07] so it should not react to higher concentrations of theophylline. So, you can just see slight variations. And if we add theophylline and we have actually the aptamer in the micro RNA you can see we have a bit higher activity of our enzyme, while we can’t reach really the, if you want wildtype levels. So, like the activity off the enzyme. 

[Image continues to show the same slide on the screen]

If we use our natural occurring micro RNA that we’ve just re-engineered a bit, you can see we have far more control. So, that’s nearly going back to full activity. We are on 80% here which is just above 100% but you clearly can’t go above 100. It’s just a variation in the measurements but we definitely have improved control over the enzyme formation.

[Image continues to show Nina inset in the top right talking and a new slide appears showing a photo of engineered tissues in petri dishes and text appears: Synthetic Biology Meets Tissue-Engineering]

So, that’s the sensor that we wanted to use to put in our tissue engineered jellyfish. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing diagrams of a Cad model being put through a bio ink process through a 3D Bioprinter and a bioprinted jellyfish in a petri dish, and then the image of the jellyfish under a microscope and a text heading appears: Construction of a pseudo-organism – 3D-Bioprinting, Design, Build, Test, Learn, Jellyfish 2.0 prototype]

So, let’s have a closer look on how we actually started making this pseudo organisms. So, it was all done with 3D-Bioprinting. You use a CAD model. You will have some hydrogel as link. We use our cardiomyocytes that we make from STEM cells. We mix it all together and we put it in the 3D bioprinter, simple enough, and print. And we’ve done that first with some fibroblast cells just to check if we can do it. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a photo of the engineered jellyfish, and the bioprinted multicellular structures after 1 hour and 1 week and text appears: Construction of a pseudo-organism, Bioink – Gelatin-methacryloyl (GelMA) & photoinitiator LAP, Cells – fibroblast cell line (3T3), Photocuring 365 nm, 3D-bioprinted multicellular structures – 3T3 cells]

So, here we used these fibroblast cells, very robust cells, and you have a particular ink, and the photo initiated, it just means you cure the material with light but it is actually not harmful to the cells and you can see these beautiful tiny constructs all in six [21:05] plates and it’s one hour after printing, a week after printing, and TSM higher magnification. So, it’s all easy enough with some of those fibroblast cells. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing a flow chart of the construction of a pseudo-organism using stem cells through the different stages and text appears: Construction of a pseudo-organism – Timing, 2 published protocols to generate cardiomyocytes, Directed differentiation – 2 weeks, Protocols closely recapitulate different stages of cardiac development, Feeder cell depletion, Day 2, hESC, Day 0, Cardia mesoderm, Day 2 – 3, Cardiac progenitors, Day 5, Cardiomyocytes, Day 10, Beating cardiomyocytes, Day 15]

But the next step was to actually use our embryonic STEM cells, or like cells that were differentiated from these embryonic STEM cells. So, timing really matters. As we all know in life that’s very true, but also in tissue engineering, it’s even more important. So, what we’ve done is we’ve followed protocols to make these cardiomyocytes, these heart cells. It’s pretty much a two week protocol and it closely recapitulates the different stages that cells would go through in our own development of our heart. So, you have your human embryonic STEM cells, then you go to cardiac mesoderm. You make so called cardiac progenitor cells. So, they are really, they can’t really go anywhere else, except downwards the way of cardiomyocytes and other heart like cells that we need. So, we started printing with our 3D printer with human embryonic STEM cells because we thought it would be really cool if we can print it and then differentiate it in the construct itself. 

Well that didn’t work. We used cardiac progenitor cells. So, that’s like Day 5 cells. So, we thought that’s probably a bit easier, the cells already kind of are forced in a certain direction of differentiation. But again, we had no luck with those particular cells. And then we ventured into cardiomyocytes using them at Day 12. So, we were a bit hesitant because these heart cells just stick together quite firmly through the tight junctions and actually disassociating them we thought we might not be able to make them beat again if you dissolved this tight junctions first. 

[Image continues to show the same slide on the main screen]

However, in the control experiments it all really worked fine. So, we took some of these heart cells, dissociated them and just replated them and they were beating fine. So, we were like, yes that’s going to be it. Let’s take those heart cells and let’s print. And we actually did it once. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing seven different photos of the bioprinted jellyfish like pseudo-organisms and text appears: 3D-bioprinted jellyfish-like pseudo-organism]

So we, we had 36 trials and we managed to get beating clusters in these jellyfish like structures only one single time. So, it’s all not that easy. But what you can see here is part of the jellyfish structure, and you see these clusters, and they’re all positive for all these heart markers, CD90, CD172, and actually we kept those cells alive for over 60 days and it was still beating. So, it’s definitely possible to keep these heart cells alive and keep them beating but what the problem was, was actually to have them nicely distributed. So, it was all clusters all over the place. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing the parts of the jellyfish-like pseudo-organism labelled and text appears: Key features – Jellyfish-like pseudo-organism, Scaffold material, biocompatible, degradable, high degree of batch to batch consistency, Cellular system, water adapted cells, jellyfish cells, jellyfish stem cell-like cells, No reproductive organs, limited lifespan, no uncontrolled spread, Structural design, mimic juvenile jellyfish…, Organism-like function, Bioremediation + luminescence, Safe, Unsafe, Directional movement]
 
And yeah, so unfortunately this was the end of the story. But you can have a vision for the future and my vision is to use some other cells, like water adapted cells for example from a jellyfish and to actually make the whole system even cooler. Like if you get this micro RNA switch in, that detoxifies and cleans up water, wouldn’t it be cool if you also have a luminescence signal so whenever this thing kind of lights up you know it’s unsafe. And like directional movement would be also really beneficial if it would just come to, and swim towards the toxin and integrate it. 

[Image continues to show Nina inset in the top right talking and a new slide appears showing USC, UQ, UNM, Genecology and CSIRO logos and a photo of a kangaroo outside the USC and a jacaranda tree at the University of Queensland and text appears: Acknowledgement, Joanne Macdonald, Bradley Harding, Justin Cooper-White, Nick Glass, Aswathi Gopalakrishnan, Darko Stefanovic, Alisha Anderson]

Yeah, and that is it. Thank you for the opportunity to share my data. I would like to acknowledge all these people, Joanne and Brad from USC, Justin, Nick and Aswathi from UQ, Darko from the University of New Mexico, and Alisha from CSIRO, and also Genecology. And clearly the funding from CSIRO and USC and Genecology. Thank you.

[Image changes to show Owain talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Owain Edwards: Thanks Nina. People if they have questions could they please put them into the Chat. Nina I have some questions. I mean I… in terms of the, in terms of the aptamer, are there any structural constraints in terms of particular targets that can and cannot be targeted using an aptamer? 

[Image changes to show Nina talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Nina Pollak: Yes, so with the aptamer that is quite tricky. So, there are lots of aptamers that are targeting, like DNA aptamers that are targeting lots of different small molecules. Like, you have 10,000 in a data base and they’re all highly selective and very specific. But in my case with the micro RNA I needed RNA aptamer and there is actually probably only this theophylline RNA aptamer that’s really suitable. We tried three different aptamers but they didn’t work for us. One was just only working like really high temperatures, like 45 degrees upwards. And so, although as a summary I would say the aptamer in this particular case is the problem, we just don’t have enough aptamers. Yeah, it would be lovely to have more of them for different components.

[Image changes to show Owain talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Owain Edwards: And in terms of the micro RNA switches, working with micro RNAs, I mean they’re impacted by an environment a lot in general and I wonder if you take it outside of a controlled laboratory setting, how functional you think they could be in a real world application if we’re putting them in to, this little organism?

[Image changes to show Nina talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Nina Pollak: Yeah, I wouldn’t be too worried about that. It’s more about proving that that micro RNA doesn’t target anything else that you don’t want to be targeted. That could be a problem. But you’re making it specific with these structural switching aptamer in it so it shouldn’t just like go over and target something else.

[Image changes to show Owain talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Owain Edwards: And so after your health project is over, which obviously was very ambitious, do you think it’s still possible to achieve this remediation pseudo-organism in the future?

[Image changes to show Nina talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Nina Pollak: I still think it’s possible. It’s definitely possibly but I think that the 3D printing technology isn’t there yet. Lots of people think we are close to printing a whole heart, like organs for personalised medicine, but we’re not there yet. It is very difficult to print larger objects due to diffusion for example. You need to get like energy resources in like oxygen and so on. It’s just like, there are lots of different issues around this whole thing but in principle I’m convinced that it can work and that it’s a beautiful way going forward because these pseudo-organisms they don’t reproduce. So, if we let them go we can probably even programme a kill switch in and kill them off if we want to.

[Image changes to show Owain talking on the main screen and participants can be seen in the participant bar at the top of the screen]

Owain Edwards: Cool, that’s what I hoped you’d say, that it is still possible. But it was always such a, the proposal was very popular. We thought, all of the Executive thought it was really interesting. I don’t see any other questions from the Chat. Are there any Louise? If not, I think we’ll move on. 

[Image changes to show Owain talking in the top right corner and a new slide can be seen on the main screen showing boomerangs at the top and text appears: Successful gene editing in the cane toad (Rhinella marina) and further developing CSIRO’s genome engineering capability, Caitlin Cooper, PhD, Health & Biosecurity]

Thank you Nina for your presentation and we’ll switch over now to Caitlin’s presentation which of course is entitled “Successful genome editing in cane toads” and a separate section, “Developing CSIRO’s genome engineering capability”. All set Caitlin? We’ve got the presentation view up.

[Image continues to show the same slide and the inset changes to show Caitlin talking in the top right corner]

Caitlin Cooper: Yep, I’m just changing the display settings right now. How’s that?

Owain Edwards: I had this problem yesterday on a Webex where we couldn’t get it… oh there we go.

Caitlin Cooper:  There we go. You’ve just got to give it a second to figure out what’s happening. Alright, thank you Owain for inviting me to speak and yes today I’m going to be talking about a project that I did in conjunction with the SynBio FSP around developing methods for gene editing in the cane toad and I’m also going to talk about a new project that we just kicked off which is a cross-business unit project in CSIRO looking at further developing CSIRO’s genome engineering capability in a strategic OneCSIRO way.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing photos of a cane toad, and some things used in the genome engineering process and text appears: Cane toad overview, Project aims, Development of protocols for breeding and genome engineering in the cane toad, Tyrosinase knockout, Bufotoxin hydrolase knockout]

So, first of all I’ll give a little bit of an overview about what I’m going to talk about with the cane toads. So, we’ll talk a little bit about the project aims and what we were trying to achieve. Then a little bit about the development of the protocols for breeding and the genome engineering of the cane toad which is really the meat of the project. And then I’ll talk about the two different lines that are generated which were Tyrosinase knockout and Bufotoxin hydrolase knockouts. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a graph on the right measuring animals that are toad naïve and toad smart, and a cartoon below and text appears: Proof of concept – Conditioned taste aversion, To discourage native predators from eating cane toads scientists feed them toad meat with emetics or very young toads, This causes them to get sick and hopefully associates that sickness with cane toads, This is more effective with young toads as it stimulates prey drive and more closely mimics what happens in the wild]

So, the proof of concept, so what we decided to look at as a main theme for the project was looking at conditioned taste aversion. So, conditioned taste aversion is a strategy to discourage native predators from eating cane toads and it’s using either very small cane toads or using cane toad meat that’s been mixed with emetics. So, basically things that make animals throw up. 

And so, how this works is you know, you feed either the small toads, or the meat with the emetics. The native predators eat that and they get sick and then they learn to associate eating cane toads with being sick. And they’ve shown that this is an effective strategy for certain species, depending on how they predate. But they’ve also shown that the small cane toads are actually a much better target trigger for this than just the meat mixed with the emetics and they think this is because actual cane toads, you know, elicit a prey response. You know, they look a lot more like a cane toad than a piece of meat does and so predators that rely more on sight, as opposed to just smell, learn a lot better with those small cane toads.

And so, the idea was can we actually replicate this in a full sized adult cane toad, make a cane toad that’s less toxic and be able to use that in conditioned taster version just as a proof of concept to demonstrate that gene editing can have an impact in the space of invasive species and be like an easy entrance into using the technology in this space.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a photo of a parotid gland on a cane toad and arrows appear creating a flow chart showing an animation of Bufotoxin linked to a sick looking snake and Bufogenin linked to a dead quoll and text appears: Deletion of bufotoxin hydrolase to generate a low-tox cane toad conditioned taste aversion]

And so to do that we were aiming to delete bufotoxin hydrolase to generate a low-tox cane toad. And so, this is a picture of a parotid gland. So, the parotid gland is where cane toads produce their toxin and so this is the inside of the parotid gland. And so, in here you’ve got bufotoxin and so that’s the storage form of this toxin. And so bufotoxin actually is less toxic. It makes animals sick but it’s not, it’s not lethal in most cases. Now, when bufotoxin is released it’s basically squeezed out those pores that are on the top of the parotid gland. There’s an enzyme known as bufotoxin hydrolase, which as you can assume it cleaves bufotoxin. And so bufotoxin is then cleaved into bufogenin. And so, on the skin, on the outside of the cane toad, it’s actually bufogenin that is present if, you know, a predator’s eating a cane toad. And so bufogenin is extremely toxic. It’s an extremely toxic cardio toxin.

[Image shows a pair of scissors cutting a DNA strand below the flow chart and then image shows the Bufogenin being removed from the slide and then the image shows a photo of a goanna looking at a cane toad and text appears: Yuk!]

And so our idea is we just take CRISPR Cas9, we go in, we delete the bufotoxin hydrolase enzyme, so you no longer get bufogenin. And so you only have bufotoxin. And so you have an animal that can make a cane toad that if eaten makes animals sick but doesn’t kill them and so, to teach them to not eat them.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing photos of male and female toad, linked to a photo of sperm, and ova, linked to show toad eggs in a petri dish, and then a fertilised eight cell egg and then a tadpole and text appears: Breeding colony]

And so, we started this off by establishing a breeding colony and then also started to get reproductive material from our breeding colony. So, we had males and females and we developed induction protocols to be able to collect sperm and ova from the males and females because to do genetic engineering it’s really important that you have access to reproductive material and also you have control of that reproductive material so you can do timed fertilisations. And so, put them together, fertilise them, and then in the end hopefully get successful fertilisation. So, you can see this is a successful fertilisation. And we’ve got an eight cell embryo there that we’re able to create in the lab which was great. 

This is actually a lot harder than we thought it was going to be because cane toads, you know, are super, you know, they’re everywhere. You know they’re a really hardy species but bring them into the lab and we have some issues but we were able to work through that which was great. And you can see we were able to generate toads. 

[Image changes to show Caitlin Cooper inset in the top right talking and a new slide appears showing a series of photos showing the development of the toads from fertilisation to adult toads and text appears: Tadpole to Toad, Day 1, Day 2, 1 week, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 6 months]

And so, it took us a while to get the right system to bring us from fertilisation, to early embryos, to actual swimming tadpoles, to metamorphs, and then all the way through the whole situation. But we’ve done that and you know, we’ve now successfully bred from animals that we bred ourselves. And so, we no longer actually have any wild caught animals in our colony. All of our colony animals are now self-generated.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing photos of toads affected by the TYR knockout showing blond patches on their bodies and a chart appears on the right and then a photo of the TYR guides 1 and 2 and text appears: Initial target: tyrosinase]

So, once we have the reproductive protocols worked out and actually knew that we could take, you know, fertilised embryos and actually raise them into toads, we were ready to start applying our CRISPR Cas9 enzymes. So, our first target was tyrosinase, which is a coloration gene. It’s a really common first target for lots of gene editing projects, just because it has a really easy to screen phenotype.

And everybody wants to make black and white animals. So, here this is just an example of a tyrosinase knockout in Xenopus. And so, you can see naturally there they really don’t colour but you can see that they have a mosaic knockout and so they’ve got blond patches all over them. And so, we were lucky, so this is actually, when we started doing this, this is before the cane toad genome was released but there were bits and pieces of genes that were available and so tyrosinase was one of those and so we were very lucky with that. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing photos of non-injected cells with gel intact, de-gelled injected cells, and then fertilised cells and text appears: Delivery via microinjection]

And so, we designed some guides, did some in vitro acids to see if they worked. They looked pretty good. And then, so we started doing direction via micro injection. And so that’s how people traditionally make genome engineered Xenopus and so we thought first port of call we would just try to replicate those activities and so we tried that. It took a little while. 

So, over here we’ve got this little embryo that looks a bit like a raisin. It took us a while to work out the protocols, you know, and Xenopus is a little bit different than the cane toad. And so, there was an iterative process, you know, that great syn-bio build, test, learn process going on to actually get this to work. But eventually we were able to do that and so we were able to do injections and get them to develop correctly which was fantastic. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a PCR and circles appear around the wildtype band from a control tadpole, a wildtype band from Cas9 tyrosinase injected tadpole, and a double cut band from Cas9 tyrosinase injected tadpole and a deletion sequence below and text appears: PCRs of tyrosinase gene from swabs taken from day 9 tadpoles injected as embryos with Cas9 protein and mRNA mixed with TYR guides (crRNA+tracrRNA) in January 2019]

And so, from here, this is a PCR of, from animals that we injected with R tyrosinase guides. And so we had two guides both targeted to Exon 1 of tyrosinase and so the idea is that if you have both guides working you should see a significant chunk taken out of your PCR and so you can see on the band, way over on the end, you have two bands there, the wildtype band, and the deletion band. And so, actually, and so that’s just a sequence of the deletion there, and then in blue you can see where our CRISPR targets are with the PAM sites in boxes and so, you know, our deletion band is lining up, our sequencing is lining up with what we would expect from the PCR.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a series of photos of tadpoles with pie graphs on the left showing the mosaicism phenotypes and text appears: Nine days old, Control tadpole, Cas9-Tyr-3 Tadpole, Cas9-Tyr-4 Tadpole, Cas9-Tyr-5 Tadpole]

And so, if we actually look at it we actually have more than one cane toad that had this mosaic phenotype which is indicating that sometimes only one of the enzymes is cutting but it’s enough to actually oblate the gene and so if you’ve one cutting or the other cutting, then you can still get this phenotype. And so you can see here we had a control tadpole, it’s all black. And then in three to four knockout tadpoles you can see that they’ve got that mosaic phenotype. And so, we assessed the mosaicism to see, you know, how many different allelic variants we had and it was, you know, it was pretty significant which indicates that the editing was happening further on in embryo development because you have lots of different allelic variants happening.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing photos of a control toad showing normal colouring, a Cas9-Tyr-4 toad, and a Cas9-Tyr-5 toad showing blond coloration and then pie graphs appear on the right showing the percentage of mosaicism and text appears: 6 months old]

And so, these are two of the animals at six months old. So, you can see they’ve got that, those blond patches on different parts of their body, especially their legs. So, that was exciting and it was great to be able to just see that phenotype. And we did some testing and basically, you know, it’s really hard to put a specific level of mosaicism on these animals because basically if you take a swab in one place versus taking a swab a different place, you get different results from the DNA. And so, it just means that their mosaicism is different depending on what tissue type you’re looking at.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a mapping diagram of the bufotoxin hydrolase gene and text appears: Bufotoxin hydrolase gene schematic, Cane toad BT hydrolase, Xenopus Tropicalis carboxylesterase 5A]

So, our next target was the bufotoxin hydrolase gene. And so, the first thing that we had to do was basically map this gene because it hadn’t been mapped before. And so, we were lucky our collaborators were able to give us the CDNA sequence and then by this point the cane toad genome had been released and so we were able to map the bufotoxin hydrolase gene. We found its most closely related cognate was a gene in Xenopus Tropicalis, which is carboxylesterase gene.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a gene guide and text appears: Region 12,500 – 16,000 in the cane toad bufotoxin hydrolase, (CTBH) gene with guides targeting regions in exons 11 and 13, Cane toad BT hydrolase]

And so then we designed guides against the androgen of the gene and we also had, you know, [40.33] and everything like that. So, we designed those guides to cut out a couple of the final exons of, of the enzyme. And we chose this because this is one of the enzymatically relevant areas of the gene. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a photo of cane toad sperm, and then a photo of the deletions in the cane toads and text appears: Delivery via sperm transfection assisted gene editing (STAGE), DAPI stained cane toad sperm transfected with Cas9 protein and a fluorescently labelled guide RNA, Sperm transfection with Cas9 protein and RNAiMax with CTH guides targeting region 12,500 – 16,000, 20 individual animals, screened with CTH 12, 5000-16,000 PCR, 1KB BT – benchtop, Promega, PC – positive wildtype control, NC – negative (no template) control]

And so for this one we decided to go with sperm transfection assisted gene editing which was, which is a method that I developed originally for use in chickens. And so, basically you’re just instead of microinjecting with a needle, what you’re doing is you’re delivering your gene editing components via sperm. So, you’re doing a sperm transfection with RMPs, so Cas9 protein and the guides who are already complexed to them. And then just, you know, using the sperm to do what it does, which is deliver things. And so that’s what we did.

So, here we’ve got a picture of some cane toad sperm. They’re stained with DAPI, so that’s the nucleuses is in blue. And then you can see we used fluorescently labelled RMPs and so that red, those red dots are the co-localised RMPs with the sperm. And so, from this we… so because the sperm, because STAGE is such a less invasive method we were getting much, much more successful hatch rates and so because you’re not stabbing, literally stabbing the embryos it’s a much gentler method and so we were getting much better hatch rates and so a lot more animals to be able to screen. And so, we got lots of animals and we got lots of animals with the deletion that we were expecting. And so, and I’ve circled a couple that actually no longer have, at least, you know, in these samples, they no longer even have a wildtype band, which is indicating we’re getting a bi-allelic knockout in the first generation which is sort of the holy grail of gene editing because it means that you don’t have to wait another generation to breed again and you’re not going to have all that mosaicism like we saw when we did our micro injections.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a diagram of the sequencing results and text appears: Sequencing results]

Sequencing results were pretty much as expected. They were pretty similar between different animals which is good. It means that the, you know, the guides are quite efficient if they’re cutting the same way in lots of different animals. Obviously, there were a few ones that were a bit different but overall they looked really good. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing photos of edited cane toads and control group cane toads]

And these are the animals. And so, that’s, that’s the project. We still have these animals going right now and we’re looking to breed these animals for next generation to do some more different experiments and we’re also planning an experiment right now where we do a head to head comparison of STAGE versus micro injection to really dig in a little bit more about the differences in mosaicism that you see and also using the same guides and using the same targets, get a real comparison of how, you know, how they work against each other. And so that’s it for that.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing text: APaIR project investigating different genome engineering platforms, Genome engineering is a strategic growth area for multiple different business units within CSIRO, The IP and licencing landscape around CRISPR/Cas9 is complex, This has created freedom to operate issues which limit CSIRO’s ability to work with commercial partners using CRISPR/Cas9, There are other genome engineering platforms with clearer IP ownership which could provide a way forwards and give CSIRO a significant strategic advantage]

The next thing that I’m going to talk about is an APaIR project within CSIRO that has been started to investigate different genome engineering platforms. And so this is a large cross-business unit project that has been undertaken. It just started in April and so myself and Tim Doran are in charge of leading the project, and Owain is one of the Co-species Leads in the project. And so, it’s a really exciting project that we think should help us move forward in the genome engineering space. 

So, genome engineering is a strategic growth area for multiple different business units within CSIRO and obviously other institutions as well. And a lot of people use CRISPR Cas9 but the IP and licencing landscape around CRISPR Cas9 is complex. There’s been some people in CSIRO that have been trying to negotiate with the BIRT Institute for instance who has underlying IP for use of CRISPR Cas9 in commercial projects, and it hasn’t really been going anywhere at this point. And so, that’s a pretty big roadblock, so that, you know, that freedom to operate issue limit CSIRO’s ability to work with commercial partners using CRISPR Cas9. And so, you know, it means we can’t really get our science out the door and having those impacts that we really want to have.

And so, the idea was that, you know, can we use other genome engineering platforms that have clear IP ownership which could provide a way forward to move forward with our commercial partnerships and also give CSIRO a strategic advantage and also, you know, provide those services to other institutions as well. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing text: Project plan, Platforms, CRISPR/Cas9, CRISPR/Cas12a (CPF1), TALONs, Mad7, Species, Saccharomyces, Pichia, Wheat, Canola, Drosophila, Mice, Quail, Zebrafish]

And so, the project plan is that we’re going to assess four different genome engineering platforms, so CRISPR Cas9 as the one that is most commonly used and is sort of our benchmark system. We’re also going to be using CRISPR Cas12 or CPF1 as another variant that people commonly use. We’re going to also try TALONs which, you know, were commonly used about five or six years ago but then CRISPR came a lot more popular. And we’re also going to try Mad7 which is a fairly new enzyme and we’re going to… it’s a CRISPR like enzyme. It uses the protein and RMT situation but it’s new and it’s different and it’s not encumbered by the same IP issues that the CRISPR Cas systems are. And so, we’re also going to be looking at this, these four platforms in eight different species. So, we’re going to be looking at saccharomyces, pichia, wheat, canola, drosophila, mice, quail, and zebrafish.   

So, these are all model species and you can see this is a broad ranging project. We’ve got people from Ag and Food, we’ve got people from Land and Water, we’ve got people from Health and Biosecurity. And so, we’re really trying to make this a co-ordinated project where we take a OneCSIRO approach to answer the question of what is the best platform that we should use moving forward both from a freedom to operate standpoint but also from a technical standpoint. And one of the biggest barriers to using these other systems is that they’re not as easily available. You know, you can go online and you can just buy CRISPR guides really easily and CRISPR protein, or CRISPR RNA, or there’s tonnes of different CRISPR plasmids available. And so, what we’re trying to do is lower that barrier and say, “OK, if all of those things were available for all of these systems, which one is actually the best?”.

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a photo of mosaicism in a toad and a DNA sequence photo and text appears: Technical questions, Which tools are most effective at cleaving DNA at a specific target site, Involves assessing efficiency, off target effects and evaluating the impact of design, Which tools are most effective at generating stable breeding lines, Which cell type is the tool delivered to? How is the tool delivered? What form of the tools works best in-vivo? What are the levels of mosaicism of somites versus gametes?]

And so, from a technical perspective, we really tried to answer two questions. First, is you know, which tools are most effective at cleaving DNA at the specific target site, which seems a pretty obvious question obviously. And so, this involves things like assessing the efficiency of the chart guides, looking at off target effects, and then evaluating the impact of the design. So, you know, if you’ve a great tool but you don’t have, you know, any good software to be able to come up with a good design that works but also doesn’t provide off targets then you’re going to have some issues. And so, it’s finding something that works really well in all of those.

And then the next question is also what tools are most effective at generating stable breeding lines. And so, this is a bit of a different question because, you know, there’s lots of ways that you can deliver genome engineering components to say like cells and culture. And so, like plasmid based systems and that sort of thing, which aren’t really applicable if you’re delivering them to embryos or in plant situations, you know, you have to use like an agrobacterium and things like that. 

So, what works well in cell culture might not work well in, in other situations and so this is, you know, what cell type is the tool being delivered to? Are you delivering it to a single cell embryo or are you delivering it to germ cells? How is the tool delivered? Is it, you know, something say like micro injection, or is it like STAGE with a transfection, or is it some other thing? What form of the tool works best in vivo? Is it a protein, is it an mRNA and then, you know, what levels of mosaicism are you seeing in both the somites? So, like the body cells versus the gametes, and the gametes are the most important because that’s what you’re going to be using to breed your next generation. And so, if you use something that works great as a plasmid in cell culture but you can’t use it to, you know, generate an actual animal then it’s not going to be the system you want to move forward with and so that’s why it’s really important that we answer both of these questions. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a project flow chart moving from co-ordinated tool design to the final report and strategic plan and text appears: Project workflow, Co-ordinated tool design, Batch plasmid and protein production, Species specific in-vitro and in-vivo testing, Pooled sequencing and data analysis, Final report and strategic plan]

For the project workflow, so we’re co-ordinating our tool design. So, we’re working with Denis Bauer’s group in Health and Biosecurity. And so they’re designing all of our tools for us which is fantastic. We’re also doing batch plasmid and protein production. So, we’re working with the SynBio Bio Foundry to produce all of the plasmids for the project. We’re also going to be using the CSIRO Protein Production Facility to produce our different proteins which is fantastic. It also means that there’s a level of quality control across the whole project.

Then for the in vitro and in vivo testing it splits off into all the different species. So, different teams are responsible for doing that within their species. Then we bring it all back together again for pooled sequencing and data analysis, which again is going to be taken on by Denis’ team. And then finally we’re going to develop a final report and a strategic outlook for how CSIRO should move forward in this space, based off of all the data that we’ve collected. And that’s it. 

[Image changes to show Caitlin inset in the top right talking and a new slide appears showing a photo of a cane toad, a photo of two cane toads mating, and a photo of a tadpole and text appears: Thank you!]

So, thank you for listening. Thank you to everybody who’s involved in both of the projects, especially the people in the [49.56] facility who take care of the cane toads. They’re fantastic. And to CSIRO and the SynBio FSP for helping fund the project. And also the CSIRO Executive for funding the APaIR genome engineering project. Thank you.

[Image changes to show Owain on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Owain Edwards: Thanks, thanks Caitlin. I’ll give people, I’ll ask some questions and give people the opportunity of putting additional questions through the Chat function. So, Caitlin, well first of all in terms of the cane toads, one of the critical questions for us is, is anybody out there interested in funding a continuation of this work because it would be such a shame if, if it just had to stop because of a lack of interested parties taking the risk of a syn bio approach?

[Image changes to show Caitlin on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Caitlin Cooper: Yeah, so we’ve actually been pretty lucky. We haven’t personally been funded to do more work but a collaborator of ours at the University of Melbourne has just gotten a grant to look at genetic bio control in the cane toad and so, you know, we’ve, we’re planning on collaborating with him and he’s really interested in, you know, taking some of the techniques and learnings and all of that sort of stuff and so we’re really interested in collaborating with him because, you know, it doesn’t really make sense to have two facilities that are making cane toads right down the street from one another. So, we think that’s a really good way forward for us.

[Image changes to show Owain on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Owain Edwards: And as somebody who’s also working in the marine space with broadcast spawners such as corals and starfish, is, is the STAGE technique a potential option for species like that? Has it been considered? Have you considered it or is there any, can you think of a barrier that might stop it being used in organisms like that?

[Image changes to show Caitlin on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Caitlin Cooper: Yeah, so we actually are considering it in a broadcast spawner, not from an invasives perspective, but from an agriculture perspective we’re looking at different fish species and so barramundi is one that we’re looking at and that is a broadcast spawner as well. And so, I don’t think that there’s a fundamental reason that STAGE couldn’t be used in that but I do think that there will be some really interesting technical issues, you know, especially around sperm harvesting and capacitation, yeah, like I don’t know how one would harvest sperm from a coral that’s not a…

[Image changes to show Owain on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Owain Edwards: Oh, it’s pretty easy. One day during the year it’s very, very easy. 

[Image changes to show Caitlin on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Caitlin Cooper: Yes, yeah and so, you know, that’s an issue and then, you know, for a lot of broadcast spawners, you know, the viability of the sperm and eggs is a very limited window and so I think that that’s, you know, that’s the biggest technical issue is just trying to figure out how you can work within that really small window of time. And then also I think, you know, separate from that is, you know, especially things like coral, the implications of salts as opposed to other cultured conditions. And so, most of the cultured conditions we’re looking at are non-salt water conditions. And so, whether you’d have to look at different suites of transfection re-agents that could work in salt water would be an added layer of difficulty onto salt-water fish and other salt water species. 

[Image changes to show Owain on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Owain Edwards: Oh, we do have a question in the Chat. In STAGE, can the CRISPR RMP cut the sperm DNA or is the DNA too bound up with protamines? If so, could you modify sperm pools to inactivate genes or shred chromosomes to bias offspring, example sex bias by x or y shredding?

[Image changes to show Caitlin on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Caitlin Cooper: Yeah, that’s a really interesting question. So, we’ve tried to answer that but it’s very difficult. What I can say is that STAGE definitely works on the maternal allele as well as the paternal allele. And so, when we first did our experiments in chickens we did cross-breeding and so we had a line of GFP chickens and so we would use STAGE on sperm from GFP roosters mated to wild type hens and then we would also do the cross. So, wild type roosters and GFP hens and we saw knockout in both of those crosses. 

So, it indicates that we can’t rule out it working in the sperm but we can definitely rule in that it’s absolutely working in the post fertilised egg. And so, but I, you know, based off of the sperm physiology I would say it’s unlikely that it’s working in the sperm because the DNA within the sperm in particular is very, very, very tightly wound. But because the sperm itself doesn’t have DNA repair enzymes it’s a really difficult question to answer because we can’t just apply it and then, you know, say do PCR and do sequencing and stuff like that because if it is cleaved then it’s not going to be repaired. It’s just going to be two pieces of DNA floating in the wind. So, yeah. But yes, we do believe that it’s actually, it’s really the sperm is just the delivery mechanism, that it’s not active in the sperm. But we haven’t proven that.

[Image changes to show Owain on the main screen talking to the camera and the participants can be seen in the participant bar at the top of the screen]

Owain Edwards: Well, I did have another question about the reach through of the Cas9 patent. I would just caution people, we’re running out of time here, there is quite a significant reach through clause in the Cas9 patent which means you can’t do research with Cas9, identify your targets etc., etc., and then switch to the TALON to generate your product. According to the patent at least, that would still be covered. There would still be a claim from the Cas9 patent if you took that approach. And so that’s another reason why it’s important that we investigate these others. So, I think we’d better close it off there. And so, I’d like to thank both speakers of course for, for their excellent presentations today. 

[Image shows Owain clapping on the main screen and then continuing to talk and the other participants can be seen in the participant bar at the top of the screen]

So, if you could give a round of applause, turn your cameras on but leaving yourself on mute and just join us with however you prefer to applaud under Zoom Webex meetings. If there are any unanswered questions they’ll be provided to the speakers for response or you can continue to put them into the Chat if you want to. Just a note that there won’t be a seminar in June. The next one is actually going to be in July and is industrial bio-technology seminar.

[Image shows Owain talking on the main screen and Louise can be seen giving the thumbs-up symbol in the participant bar at the top of the screen]

So, Louise is that everything, have we covered everything? Yes I get a thumbs-up which is very unusual for me. 

[Image shows Owain waving and then continuing to talk and Louise can be seen waving in the participant bar at the top of the screen]

So, thanks everyone for participating and enjoy the rest of your day and thanks again Nina and Caitlin. Fantastic.

[Images move through to show an O on the screen, Louise listening and then Louise’s photo on the main screen and the participant bar can be seen at the top of the screen]