March-2021-SynBio

SynBio FSP Monthly Seminar Series

[Image appears of Dr Kristie Jenkins on the main screen talking to the camera and participants can be seen in the participant pane at the top of the screen]

[Image changes to show a slide showing a photo of the Australian Centre for Disease Preparedness research facility and Kristie can be seen inset in the top right talking and text appears: Platform Z, Developing the chicken egg as a medical device, Tim Doran, Mark Woodcock, Mark Tizard, Kristie Jenkins and Andy Bean, CSIRO Health & Biosecurity]

Dr Kristie Jenkins: This meeting’s going to be recorded and will be available on the FSP website. So, thanks everyone for joining us for another FSP seminar. I’d like to begin by acknowledging the Wathaurong and the Kaurna people, who are the traditional owners of the land where Tim, Fatwa, and myself are today, and pay my respects to their Elders past, present and emerging. I’d also like to extend an acknowledgement to the traditional owners of many of the lands of which people are joining this meeting from. 

So, for those of you who haven’t joined one of these before, all the questions just go in the Chat function and then we’ll read them out after each speaker. If we get too many questions and we can’t cover them all off we’ll make sure that those questions are forwarded to the speakers and make sure that they get answered for you. If everyone can stay on mute when they’re not talking that would be great. I think that’s all the things I need to cover off on. 

So, just before we start, for those of you who don’t know, I’m Kristie Jenkins. So, I’m the Health and Medicine Domain Leader within the FSP. And today we have two speakers. So, we have Tim Doran, who’s based at CSIRO, and he’ll be talking to us about Platform Z, Developing the Chicken As a Medical Device. And we have Fatwa Adikusuma, who is based at Adelaide Uni, and he’s one of the FSP Fellows. And he’ll be talking to us about Developing Gene, Genomic Cutting and Precise Editing for CRISPR Technology. So, they’re our speakers. And we’re kicking off with Tim. So, over to you Tim.

[Image continues to show the same slide on the screen and Tim Doran can be seen inset in the slide at the top right talking to the camera]

Dr Tim Doran: Thanks Kristie. Just thumbs up if you can hear me OK. Awesome. Thank you. Well, thanks everyone for dialling in and listening to my presentation and thanks to the FSP for the opportunity to give you all an update on our Platform Z project. Just want to acknowledge everyone involved in this project. So, Mark Woodcock is really the main person that conducts this work for us. Mark Tizard, Kristie Jenkins, and Andy Bean are all involved in helping with oversight of the project. We’re based in Geelong, at the Australian Centre for Disease Preparedness, which is that big lab in the picture there. So, I can’t get the slide to go… there we go. 

[Image changes to show a new slide showing a photo of chickens in an egg hatching facility and text appears: Selectively hatching female chicks – a high priority for the global egg industry, 7 billion male chicks culled per year globally, 23 million in Australia. Welfare is acceptable, Ethics is the problem, Also… Wasteful, Labor intensive, Only 93% accurate]

So, the project that I want to give you an update on, it’s using synthetic biology to solve a big problem in animal agriculture but it’s also taking that problem and turning it into an opportunity, also using synthetic biology. So, there’s kind of two stories to tell about this one project. The problem that we’re working on in terms of the animal agriculture problem is in the egg laying industry. And that’s the culling of male chicks. So, obviously males don’t lay eggs. In the, in the global poultry industry, chickens are bred for purpose. So, the breeds that are there for egg laying are not very good at putting on, on meat. 

So, it’s not a sustainable solution to grow out the males for food production via meat. So, they’re culled at one day of age. So, the extent of that problem is about 7 billion male chicks are culled per year globally in the global layer industry. And about, that sort of equals about 23 million culled each year in Australia. The male chicks are euthanased in a welfare acceptable fashion but the industry accepts that there’s an ethical problem around the process that they would like to eliminate. And there are certain countries that are leading the way in terms of stopping the practice of culling males, and certainly some European countries are well and truly down the path of stopping the practice. And there are indications here in Australia that this, that the industry is looking to find a solution to this problem.

[Image changes to show a new slide on the main screen and Tim can be seen inset talking in the top right corner of the screen and text appears on the slide: Selectively hatching female chicks – a high priority for the global egg industry, Challenge – develop a technology to detect and remove male embryos prior to hatch, At point of lay, Without physically penetrating the shell, High through-put, high accuracy, Low cost, Global industry demand for a solution – CSIRO has major engagements with industry in US, Europe, Asia and Australia]

So we’ve… there we go… so we’ve been working on this problem for a little while. Just to define the challenge – so when you talk to industry they’d like a technology to detect and remove male embryos prior to hatch. So, as soon as the egg is laid preferably, without any physical penetration of the shell. So, they don’t want to open up the shell and then take any samples or do any imaging of the embryo itself. It needs to be a high through-put process, and it needs to have a high level of accuracy, and it needs to be cheap. So, it’s a pretty challenging set of, of priorities that we need to kind of hit here for industry. There’s a global industry demand for this solution as I’ve mentioned and CSIRO has a lot of engagements with industry right across the globe, including the US, Europe, Australia as well to try and solve this problem.

[Image changes to show a new slide showing a flow chart showing a hen and a rooster, two eggs, and one chicken, and text appears: “Null segregation” – offspring are not GMOs, Gene Technology Regulations – Amendments (for clarification of definitions), Null-segregant offspring of GMO parents will not be classified as GMOs, No culling of day old male chicks, Repurposing (value adding) of male eggs, Null-segregant process also being recognised (and excluded from GMO definition) by Food Standards Australia New Zealand (FSANZ)]

As I said, we’ve come up with a synthetic biology approach to this and this slide hopefully will explain it. And there’s a few other things on this slide I need to go through too, and it’s around the regulation of the technology that we’ve been developing as well and how it will be seen by important regulators. 

So, I’ve got my pointer on here. We’ve got a hen and a rooster and what we do is we add a fluorescent marker gene to one of the sex chromosomes of the female breeder. Now in chickens, as in all birds, the sex chromosomes are not X and Y’s as they are in mammals or in us. They’re Z’s and W’s and it’s because the sex chromosome, they differ in birds as they do in mammals. So, it’s the males that are the homogametic sex in chickens and they have two Z chromosomes. And it’s the female that’s heterogametic and she has a Z chromosome and a W chromosome. 

And so, our idea was to place a fluorescent protein marker gene on the Z sex chromosome of the female breeder, and when you mate that female breeder with a male, only the male offspring ever inherit that marker gene. So, they inherit obviously one single Z chromosome from the female, and another from the male. And so, as long as the female one is marked, the male embryos will contain a marker gene. The important thing around this technology is that the females, which are the ones which we want to go on to hatch, and then provide to industry to lay eggs, never inherit that marked chromosome. 

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

And also what we are looking to do, and I’ll go into this a little bit later, is once we’re able to select those males, how we can repurpose them and add value to those male eggs before they are ever put into incubators and hatched out.

So, Mark Tizard, who’s one of the key leads on this project has worked very closely with regulators around how this technology would be, would be seen. And the main regulator that we’ve been working with is the OTTR, and they’ve recently amended their gene technology regulations, so that null segregate offspring of GMO parents, which is what we’re generating here, will not be classified as GMOs. So, as we take the females through to hatch and introduce them into the layer industry in Australia, they would not be seen as GM. 

And the same goes with Food Standards Australia and New Zealand, who are the regulators of our food products. They’re going the same way and recognising that the null segregant process that results in an animal that’s used for food production, that’s come from GMO parents, but the marker has been segregated, they will also not be GM. So, this is really important because this helps us to be able to see how this technology can be adopted through industry and removes a major hurdle for industry in terms of adoption.

[Image changes to show a new slide on the main screen showing a photo of cells under a microscope and some day-old chickens and Tim can be seen talking inset in the top right and text appears on the slide: Primordial Germ Cells (PGCs), Avian embryonic PGCs migrate through the vasculature on their path to the gonad where they become the sperm or ova producing cells, This unique feature of avian PGC migration through blood has led to a transformation advance in the generation of genetically engineered chickens, PGCs “OUTSIDE” – highly skilled culture (van de Lavoir et al, 2006), establish PGC cultures, Introduce genetic modifications into cultured cells, expand the modified cells into clonal populations, inject selected cells into recipient embryos to create germline chimeras, PGCs “INSIDE” – in vivo transfection of PGCs (Tyack et al, 2014), direct injection – no selection step available which can lower success rate, Avoid imported biologicals required for PGC culture system]

So, a little bit about the science. The cells that we work with in chickens to generate genetically modified birds are called Primordial Germ Cells, or PGCs and are essentially stem cells that differentiate into either sperm or ova in a mature male or mature female chicken. And there’s a unique feature around Primordial Germ Cells in birds compared to other animals like mammals, and that is that they circulate through the blood of very early staged embryos. So, you can get at them. And this has provided a transformational advance in how we generate genetically engineered chickens. 

And so, you can harvest these germ cells from an early stage embryo and work with them in  culture in the lab, where they can be modified, then expanded into clonal populations, and then injected back into recipient embryos to create genetically modified chickens. Or, you can actually modify these cells directly in an early stage embryo while they’re circulating in the blood. And this is the basis of the direct injection technology that our lab in CSIRO pioneered, and has patented, and has done a lot of work on to, to be able to create a whole range of chickens that have had their genomes engineered in one way or another. So, we work on this method. 

[Image changes to show a new slide showing a flow chart showing photos of a fertilised egg at 2.5 days old, hands holding an egg and sealing a small hole, a mature rooster, a semen graph, a day old chick, and then photos of a fertilised female egg yolk, a fertilised fluorescent male egg yolk, and a small video of the resulting chickens, the female chick dull and the male chick glowing fluorescently and the image shows the pointer moving along the various stages as he explains it]
 
This is kind of what it looks like for the whole sex selection process. This is something that we’ve gone through can I just move that up there. Yeah, OK, so working from left to right. This is a very early stage chicken embryo. It’s about Day 2.5 of embryo genesis in incubation. The germ cells are circulating in the blood at this stage. You can see the vasculature. This is the head up the top here and this is a micro-capillary in which we’re injecting genome engineering tools into the blood system of this embryo.

Once we’ve done the injection we seal the egg back up. So, we’ve created a little hole at the top to access the embryo. We can seal that back up, put that back in the incubator and these embryos develop all the way through to hatch. Once they’re hatched we keep males. We keep males because you can mate one male with up to ten females. So, it’s a really efficient way of being able to generate genetically modified offspring. So, we only keep the males. 

Once they’ve reached sexual maturity like this rooster here, we take semen samples, and we analyse that semen for, to determine how chimeric they are, which means how many of their sex cells, how many of their sperm cells carry the modification that we’ve created. So, they’re chimeric at that stage. And we choose those that are the most chimeric. And then we mate them with wild type females and then we screen for the G1 offspring that now contain the modification in a germline fashion, so all their cells contain the modification. So we, we hatch out chicks. We take a little blood sample and we do a PCR to look for that modification in all their cells. Down the bottom here we’re now going to go from right to left. So, here I have to get out of the pointer just to start the video. 

[Image shows the cursor making selections to turn the pointer off in an inset drop down box and then the box disappears]

How do I get out of that? Pointer options, turn it off. Can you see the video going OK Kristie, thumbs up? Yeah, OK. Right.

So, here we go. We’ve got two chicks here that we’ve hatched out. The one on the left clearly contains a fluorescent marker gene. It’s the green fluorescent protein. She’s a female and she contains that marker gene on her Z chromosome. So, all of her cells fluoresce. The little guy on the right is a male. He doesn’t contain the marked chromosome at all. And you can see, we’re taking this image using an iPhone with a filter and a UV light source. When we turned the light source off like just before then, you obviously can’t tell any difference between the males and females. So, you need a specific UV light source and a filter to be able to detect fluorescence. 

So, once this, once this female – she’s about two weeks of age – once she is sexually matured we then put her in a proof of concept mating with a wild type male and check the fertilised eggs for the presence of the marker. And I’ll just turn the pointer back on. And this image here on the right contains a male embryo. So, when a fertilised egg is laid, so this is at point of lay, we’ve cracked open the egg. You can see the yolk and on top of the yolk sits the blastoderm. 

And so, the embryo starts to develop as the egg comes down the oviduct and is laid. And at point of lay there’s about 60,000 cells. And you can see this nice ball of fluorescent light that says that this embryo is male. And over here on the left you can’t see the fluorescence. You can see the little opaque blastodisc on top and that embryo would be female. So, it shows that the technology works so… and we know that we can detect that ball of light using lasers through the shells. And we’re working now on a technology to be able to do that kind of detection in a high throughput manner, in a way that will integrate well with industry. But it shows that the whole technology works.

[Image changes to show a new slide showing a flow chart showing symbols of genetic modification of genes, a torch shining on two eggs, a mature chicken, a barn, eggs on supermarket shelves, and an egg on a plate and text heading appears above and text appears on the flow chart: Sex selection – benefiting the entire supply, Sex selection, Marker removed with male eggs, No more culling of day old male chicks, Global Genetics Companies, Nation-wide breeders and hatcheries, Local Farmers, Local Supermarkets, Local consumers, Hens to farm exactly the same as they are today, eggs on the shelves exactly the same as they are today, Nothing changes for the consumer]

And so what this slide really points out is kind of how it works from an industry point of view and it shows the supply chain from left to right. So, we need to work with global genetics companies to introduce the marked gene into their female breeders. They provide breeding stock to nationwide breeders and hatcheries. They’re the ones that have the issue with sex selection so they’re the ones that would adopt the technology where we can identify the males at point of lay, before they incubate them and take them through to hatch. Once the hens hatch out they then provide them to local farmers that farm the eggs and provide the eggs to the local supermarkets, and onto consumers. And as I mentioned these are, these eggs that come from these hens are exactly the same as they are today, the eggs on the shelves, they layer exactly the same as they are today. Just let me get this out of the way so you can see a little bit better. So, nothing changes for the consumer but we’ve been able to remove those male eggs because of the marker and it prevents the culling of male chicks. So, that’s the whole concept of the sex selection technology.  

[Image changes to show an inset on the slide showing a photo of two chickens and two eggs and text appears on the inset box: Public perceptions of using synthetic biology to prevent the culling of male chicks, Synthetic biology technologies such as gene marking could eliminate the need for culling male chicks in the egg-laying industry]

We were really, really thankful that the FSP were able to focus on this project with the help of the wonderful Social Science Team, that Aditi and Eloise look after within the FSP and take this out to the public to get their perceptions on how they perceive using synthetic biology to prevent the culling of male chicks. And there was overall a willingness from the community to see this technology adopted. So, it was a great collaboration between our team and the Social Science Team within the FSP for which we’re really grateful for.

[Image shows the inset box disappearing and then new symbols appearing at the bottom of the slide showing symbols linked by arrows of a male fertilised egg, a vaccine production facility building, and vaccines, and text appears: Platform Z Eggs, Vaccine Production Facilities, Improved Vaccines + Novel Vaccines] 

And then, this is where the next aspect of the synthetic biology story comes together and that’s seeing that those male eggs that we segregate away, you know, they can be used for low value products in the food chain. But we saw it as an opportunity to use synthetic biology to say, what can we do, what can we also add to that marker gene that’s going to add value to that egg and use it for a much higher value opportunity. So, how can we convert it into a synthetic biology opportunity? 

And we call that Platform Z, which is, you know, using the Z chromosome as a platform to introduce new traits into the developing embryo. The one that we focussed on here in this slide is around vaccine production. So, I would imagine most of you are aware that a whole range of different vaccines, including influenza vaccines, are grown predominantly in fertilised chicken eggs. So, some of the big vaccine manufacturers like say Sanofi Pasteur, who are based in the US, they use almost 2 million fertilised chicken eggs a day, every day, all year to make flu vaccine.

Here in Australia we have Seqirus, based in Victoria, they use close to half a million fertilised chicken eggs a day to make the flu shots that we get. So, we thought it might be an opportunity to say how can we improve the eggs for flu vaccine production, as an example using synthetic biology, and converting this kind of, you know, low value, almost waste stream into a viable opportunity.

[Image changes to show a new slide showing a photo of a researcher working with trays of eggs, and a photo of a researcher working with gene technology and text appears: Platform Z bio-brick development…. So far, Value-adding products for flu vaccines, SIAT1, Antivirals, Nanobodies, Genetic componentry for sex selection and detection in fertilised eggs, GFP vs RFP (mScarlet), But just the beginning…. What else can we reprogram the egg to do?, Oviduct expression and purification from egg white, How do we create a new bio-industry?]

So, Platform Z, the different sorts of bio-bricks that we’ve looked at so far include how we can improve flu vaccine production. And that’s the story I want to tell now, it’s about the SIAT1 gene. We’re looking at another opport… a number of other opportunities for using the eggs for a high-value opportunity that I won’t talk about today. We’ve also been looking at the genetic componentry for sex selection, and we started of with GFP in the example that I showed earlier. But we’ve moved on to a red fluorescent protein, namely (mScarlet). That’s a better choice for being able to detect the embryo as it develops within the egg, and particularly at the early point of lay stage. 

And we, you know, with this project we think we’re just at the beginning. You know, what else can we reprogramme the egg to do? There’s other groups out there that already use the egg as a bio-reactor to express proteins in the oviduct, and to purify from egg white. So, you know, we can link into opportunities there. And we’re also looking very carefully at how we can convert the whole opportunity into a new bio-industry here in Australia, and that’s another story as well.

[Image changes to show a new slide showing a photo of a hand holding a syringe and injecting an egg and a flow chart and Tim can be seen in the right corner inset talking and text appears on the slide: Egg adaption of the ‘flu virus vaccine – the role of SIAT, ‘flu virus attaches and infects host through glycoprotein receptors, In humans these are mostly a2,6-linked sialic acids, In chickens these are mostly a2,3-linked sialic acids, This difference leads to mutation of the ‘flu’s own receptor – “egg adaption”, Overall vaccine effectiveness against flu was 40% and 38% in 2016 and 2018 flu seasons – prevalence of egg-adapted strains, The SIAT1 enzyme can address mismatching problems, Converting a2,3-linked sialic acids to a2,6]

So I want to tell you a little bit about SIAT1. There’s all sorts of things over my screen so I’m not sure if you can see it all. But basically it’s a pretty quick story. So, when we were talking to flu vaccine manufacturers about how we might be able to use this technology to help them they told us about a problem they have called egg adaption. And that’s linked to the receptor that flu viruses use in cells. So, in mammals, and that flu receptor is called sialic acid, in mammals, the predominant form of that sialic acid is called alpha 2,6-linked sialic acid. However, in chickens, and in particular in the chicken embryo, where the flu virus is grown it’s alpha 2,3-linked sialic acid. 

So what happens, if you take a mammalian influenza strain that you want to include in a seasonal vaccine and put it into a chicken egg, the receptor modifies itself so that it adapts to the chicken form of the receptor and it’s called egg adaption and it means that the vaccine is no longer as immunogenic as you’d like because this major antigen has changed to become egg like as opposed to human like. So, when you vaccinate humans with that vaccine, it’s not exactly in the same format that you would like it to be. And in fact, that issue around egg adaption was a major problem in 2016 and 2018 in the flu vaccines that were, that were in circulation in those years. And there was only about 40% to 38% efficiency of the whole flu vaccine those years which was a really big problem for the vaccine manufacturers. What we know is that you can convert alpha 2,3 sialic acid to alpha 2,6 with a single gene called SIAT1. So, it’s a transferase gene that does that conversion. So, our idea was to introduce a construct with the fluorescent protein marker gene that can express the SIAT1 transferase, and in the embryo convert the 2,3 sialic acid to 2,6. And that would solve the problem.

[Image changes to show a new slide showing a chart explaining the process of the expression of SIAT1 in chicken cells below the text heading: Expressing SIAT1 in chicken cells makes their receptor more… ]

So, this is the project that Mark Woodcock’s being working really a lot on and he showed that once he’d created that construct and put it into chicken cells that the, here we’ve got wild type cells at the top where we’re looking at staining of alpha 2,3 sialic acid compared to alpha 2,6 and you can see predominantly that you’re getting alpha 2,3. But when you introduce the construct that overexpresses SIAT1 into these cells, you now change the profile so that you’re getting much more alpha 2,6. So, it shows that the construct’s working. 

[Image changes to show a new slide showing photos of a developing chicken embryo at a couple of days, an egg being injected, a rooster, a semen chart, a video of a chicken with mScarlet and SIAT1 and one without, chicken embryos, and an egg being syringed below the text heading: The high value protein coding bio-brick SIAT1]

And then we took that and engineered a chicken to create a transgenic chicken that now has the mScarlet fluorescent protein marker gene plus SIAT1. And so again, you know, going from left to right we did the… to do this we did the injection, the direct injection process of the construct into an early stage embryo, sealed it up, took it out to hatch, kept the males, raised them to sexual maturity, screened the males that had, that were the most chimeric for this construct in their sperm cells and then we started hatching chicks until we identified – there must be another way of doing this without having to get out of the pointer option – sorry about that. It’s going to take me a little while to get out of it. 

We identified this chick, who we called Miss Scarlet for obvious reason. So, she… let me turn the volume down, so she contains in all of her cells the mScarlet reported gene, which means that she fluoresces scarlet, again using… start that again, using goggles and a UV light to be able to identify the fluorescence. Now we’ve got the light source off and you can’t see any difference between her and the non-transgenic there on the right. But she also expresses SIAT1. And so, she’s now at sexual maturity. We’re getting, we’re getting fertilised embryos from her and this is, I’ll just get the marker gene, the pointer back on again, and in this image here we’re showing some embryos that we’ve taken out of their shells. You can see that we’re getting definite fluorescence of the mScarlet reported gene.

Mark’s done a lot of work to characterise SIAT expression and we’re now doing the detailed work of being able to show that the 2,3 is being converted to 2,6 through the staining process. And we’re now about to send these embryos to Seqirus, who we’re partnering with on this part of the project, to show that we can solve the problem of egg adaption. So, they’ll use those eggs to grow a subset of flu vaccine strains up in, and then sequence those strains to show that the haemagglutinin protein that binds to the receptor is not modifying in these eggs. So, we’re really excited that we’ve got to this stage of the project and we can’t wait to see those results back in, actually, the coming weeks. So, we’re getting really close.

[Image changes to show a new slide showing a photo of the genome engineering team posing for the camera and Tim can be seen inset talking in the top right and text appears: Thank you SynBio FSP, Genome Engineering Team, Arjun Challagulla, Caitlin Cooper, Tim Doran, Kristie Jenkins, Keilly Kuykhoven, Kirsten Morris, Kiran Krishnankutty Nair, Terri O’Neil, Suning Shi, Agus Sunarto, Terry Wise, Mark Woodcock]

So, I’ve just noticed that I’m pretty close to running out of time. I just want to, I’m not sure if you can see my big thank you SynBio FSP up the top there. It might be behind a few things. But just to say thank you so much to the Synthetic Biology FSP for supporting this project. You know, we think it’s a really, a really good project that shows how you can use synthetic biology to solve a big problem in animal agriculture to make the whole process much more sustainable, but to take an opportunity out of that process to build new, to build new, to build new opportunities for synthetic biology by converting a waste stream into a high value product like, you know, improved eggs for vaccine production.

Mark Woodcock is… here is Mark just on the right hand side of this image. You know, he’s been fantastic on this project but he’ll be the first to acknowledge that when you’re making genetically modified chickens it requires a whole team of people to help do that and I just want to thank everybody on this list here of staff within the team that Kristie leads that’s been able to help us to get to this point of the project. And with that, I’d just like to say thank you, and happy to answer questions whenever the, whenever you’d like me to do that Kristie.

[Image changes to show Kristie inset talking in the top right of the slide]

Kristi Jenkins: Thanks Tim. Don’t forget if you’ve got any questions, just post them in the Chat. 

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

I’ve got a couple. So, you mentioned about using platforms there for egg adaption. Is there any other opportunities in the vaccine field as well?

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

Tim Doran: Yeah, so that’s a really good question. 

[Image changes to show Kristie on the main screen listening and then the image changes to show Tim on the main screen talking to the camera and the participants can be seen inset at the top]

So, we have another project, I’m not sure if Andy Bean’s on the line, but that we call VaXIMISER, where we’re working on engineering eggs to improve the yield of the vaccine strains that we get in eggs. So, we see that we could also include different types of bio-bricks if you like on the Z chromosome that can target some of these VaXIMISER genes. So basically what we want to do is knock down impression of certain immune genes in the chickens so that they’re a little bit more susceptible to the vaccine strains so that the embryos can grow those viruses up to a higher level. So, that’s another opportunity that we see. So, you could end up with, you know, a single egg, where you can get a higher titer of vaccine virus and prevent egg adaption at the same time. So, you know, I think we can definitely take it another step. It’s a good question.

[Image changes to show Kristie on the main screen talking to the camera and Tim can be seen listening in the participant pane at the top of the screen]

Kristie Jenkins: Um, got another one here. How can industry detect the fluorescence at scale?

[Image changes to show Tim talking on the main screen and Kristie can be seen listening in the participant pane at the top of the screen]

Tim Doran: Can you repeat that one sorry Kristie?

[Image changes to show Kristie on the main screen talking to the camera and the Tim can be seen listening in the participant pane at the top of the screen]

Kristie Jenkins: Sorry, how will industry detect the fluorescence at scale?

[Image changes to show Tim talking on the main screen and Kristie can be seen listening in the participant pane at the top of the screen]

Tim Doran: Yeah, so that’s a really good question and that’s the next phase of the project. So we need to start working with experts on lasers and UV light detection. But, you know, the industry is already set up in a high throughput way to analyse certain components of fertilised eggs through the process. You know, so they look at egg shell integrity. They look for cracks and so on and contamination and things like that. So we see a way of being able to introduce an extra bit of kit to that process where you can detect fluorescence. It needs to be obviously at point of lay before incubation, remove the males out and then the female ones go into incubation. So, lots of discussions with industry about how it could integrate and we see that it’s possible, yeah.

[Image changes to show Kristie on the main screen talking to the camera and Tim can be seen listening in the participant pane at the top of the screen]

Kristie Jenkins: Also, and one more. Do chickens have Z chromosome inactivation mechanisms that are similar to the X chromosome inactivation in mammals?

[Image changes to show Tim talking on the main screen and Kristie can be seen listening in the participant pane at the top of the screen]

Tim Doran: Yeah, so they don’t which is really good when it comes to this process. So yeah we’ve obviously done a lot of work around not just sex selection but understanding how sex determination works in birds. And it’s actually a dosage process as Paul has kind of suggested there but it’s not about inactivation. It’s about having two copies of a particular male sex determination gene called DMRT1 as opposed to an activating one. So, that’s a really good question. I’m not sure who Paul T is but I’m guessing it maybe it called be Paul Thomas who knows a little bit about this. Yeah, he leads the group that discovered the DMRT1 gene and did that work really well. 

[Image shows Tim waving on the main screen and continuing to talk]

So, hi Paul.

[Image changes to show Kristie on the main screen talking to the camera and Tim can be seen listening in the participant pane at the top of the screen]

Kristie Jenkins: OK, I don’t think we’ve got any more questions for you at this point in time Tim. So, thank you very much and we’ll give you a thank you at the end when we give Fatwa one as well. And we’ll throw over to you if you can share your screen. It’s thinking about it. Sometimes it takes a minute. 

[Image shows a new slide appearing on the main screen showing a photo of a DNA strand, an inset photo of Prof Paul Thomas, and the University of Adelaide and CSIRO logos and text appears on the slide: CRISPR Gene Editing, Dr Fatwa Adikusuma, CSIRO SynBio FSP Fellow, Genome Editing Laboratory, Adelaide Medical School, University of Adelaide, SAHMRI]

You can see it. It’s just not in presentation mode. Perfect thank you. Over to you.

[Image changes to show a new slide on the main screen showing a diagram of genome editing and Kristie can be seen inset talking in the top right and then the image changes back to the previous slide]

Yep perfect. 

[Image shows the same slide and Fatwa Adikusuma can be seen inset in the top right talking to the camera]

Fatwa Adikusuma: Yep. Alright can you hear me now? Good, OK cool. Hello everyone. Thanks to CSIRO FSP for this opportunity. I’m working in genome editing lab with Paul Thomas. This is Paul Thomas. If you are wondering, yes Paul asked that question to Tim. We do a lot of genome editing experiments using CRISPR in order to improve gene editing performance as well as applying the technology for disease therapeutic.

[Image changes to show a new slide showing a diagram of genome editing and Fatwa can be seen inset on the right talking and text heading and text appears on the slide:  CRISPR technology for Genome Editing, CRISPR technology: RNA-guided endonuclease for site specific DNA break , 1. Cas9 endonuclease to cleave DNA, 2. Guide RNA to giving instruction to Cas9 where to cut]

So a brief background of CRISPR technology. I’m pretty sure you are all already familiar with this. CRISPR technique is an RNA-guided endonuclease that allows us to create site specific DNA cleavage. It has two components, the Cas9 endonuclease that cuts the DNA, and the gRNA that provides the instruction to Cas9 where to cut the DNA. 

So, in every cleavage the DNA target sequence should match the gRNA sequence and the downstream sequence has to contain specific sequence called PAM. For the commonly used CRISPR system the streptococcus pyogenes or better known as SpCas9, the PAM sequence is NGG. Hang on a second I’m going to need to turn on my laser pointer. 

Kristie Jenkins: Fatwa can you just move, there’s a check-in box on your screen as well over your presentation, that’s sitting over like DNA break at the moment. If you could just?

Fatwa Adikusuma: This one?

Kristie Jenkins: Yep. Awesome. Thank you.

[Image changes to show a new slide showing a diagram explaining insertions and deletions in DNA and Fatwa can be seen inset in the top right talking and a text heading appears: DNA cleaved cellular DNA repair machinery]

Fatwa Adikusuma: So, one Cas9 enzyme cleave the DNA. This will trigger the cellular DNA repair machinery to repair the break. However the repair is most of the time not perfect and will introduce mutations around the break. The mutations could be insertions or deletions, or InDels, in short. Here is the example of InDels and if you provide, yep, and if you provide a DNA template during the process, during the repair, there is a chance that your DNA template gets integrated or copied through a process called Homology Directed Repair or HDR. So, you get a precise editing. However, this HDR process is very inefficient.

[Image changes to show a new slide showing a flow chart from the cell to the NGS Analysis, a photo of a neon nucleofection, and text heading and text: Genome editing using CRISPR-SpCas9, Cells – Hek293T, R1Mes, C2C12, CRISPR nucleofection PX459.V2, Puromycin selection, Harvest cells, Extract pooled gDNA, NGS, NGS analysis using Crispresso]

We have done a lot of genome editing experiments using SpCas9 in Hek cells, mouse ES cells, and C2C12 mouse myoblast cells. When we did genome editing we used PX459.V2 plasmid for the expression of the CRISPR components, the gRNA and the Cas9. This plasmid expressed Cas9 linked to a puro resident gene, the T2A self-cleaving peptide, and its expressed under CbH promoter, while the gRNA is expressed under U6 promoter. We use nucleofections to transfect the plasmid to the cells, and since the plasmid expressed puromycin resistant gene we also performed puromycin selections to select for transfected cells. For the editing analysis we used NGS and analysed the NGS data using Crispresso tool.

[Image changes to show a new slide showing three bar graphs showing the efficiency of the genome editing and Fatwa can be seen in the top right inset talking and a text heading appears: SpCas9 creates genome editing with near-perfect efficiency]

In this study we detected 53 different gRNAs, nine in HEK cells, 28 in mouse ES cells and 16 in C2C12 cells. These graphs shows the editing efficiency of our experiments. As you can see we got very high editing efficiency, even efficiency is close to 100%. However, we found some gRNAs that we think are less efficient compared to the others. One is here, while the other gRNAs gave nearly 100 editing efficiency, this one here, gRNA mPrl 1.4 gave only 93% cutting efficiency. I know that 93 editing efficiency is very high but in our case it is unusual. In C2C12 experiments there are four gRNAs with efficiency less than 90%. Their efficiency is between 83% to 89% and we wondered what is causing this less efficient cutting activity in those five gRNAs. 

[Image shows a table appearing showing a description of the less efficient gRNAs in the bottom right of the slide]

And we suspect that the expression of the gRNA transcript is low in those five gRNAs. This is based on our observation that the gRNA sequence contains T rich sequences. And string of T6 and string of C signals for transcriptional stop for gRNA polymers three, which is the key player of transcription when using U6 promoter.

[Image changes to show a new slide showing a diagram explaining the Quantification of gRNA transcripts and Fatwa can be seen inset in the top right talking and text heading appears: Quantification of gRNA transcripts]

The proof that we quantified the gRNA transcript expression level from three less efficient gRNAs, it looks like the gRNA transcripts is lower in those less efficient gRNAs when compared to an efficient gRNA.

[Image changes to show a new slide showing a diagram explaining the PDG.V2 construct and text appears: gRNA expression from dial U6-gRNA cassettes lead to increased editing efficiency, PLOS ONE, Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression]

We thought, “Let’s try to increase the gRNA transcript level and see whether editing efficiency was getting better in those less efficient gRNAs. And to increase the gRNA transcript we initially used our published dual-gRNA construct. This dual-gRNA construct is very similar to the PX459V2 plasmid that I used before. However this dual-gRNA plasmid has two U6-gRNA cassettes. So, you can express the same gRNA from two U6-gRNA cassettes.

[Image shows three diagrams appearing on the bottom of the slide showing the gRNA scaffold levels]

We initially checked the gRNA transcript level, indeed gRNA transcript level is higher when using dual gRNA plasmid and it is consistent across the gRNA tested.

[Image changes to show a new slide showing a bar graph showing editing efficiency from dual U6-gRNA cassettes and text heading appears above: gRNA expression from dual U6-gRNA cassettes lead to increased editing efficiency]

When we used dual-gRNA plasmid, we observed better cutting activity of those less efficient gRNAs. This blue here is the efficiency using PX459.V2 and the green is the efficiency using dual-gRNA plasmid. This confirms that less efficient cutting activity of those less active gRNA was caused by insufficient gRNA transcripts. And increasing the transcript could improve the editing activity. 

[Image changes to show a new slide showing a paper on the Delineation of Exact Transcription Termination Signal for Type 3 Polymerase III and diagrams can be seen below the heading]

And we found this interesting paper. The message of this study is if you express RNA from U6 promoter a string of three or less T doesn’t stop the transcriptions, while a string of five or more of Ts will stop the transcriptions. And interestingly a string of four, four Ts will significantly reduce the transcription. And we noticed that the gRNA contains a string of T in the gRNA scaffold which means so far we express the gRNA non-optimally. 

[Image changes to show another paper with various diagrams showing how to achieve higher transcript levels below the text heading: A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System]

Another interesting study showed that we can replace one of the T and we could get higher expression of the gRNA. So, in this study they showed that they replaced the T, one of T with C and then showed that the gRNA transcript level is a lot higher. So, we took action and then modified the plasmid, PX459.V2 and to replace the T with a C, and create a plasmid that we call PX459.V3.

[Image changes to show a new slide showing three diagrams showing transcript levels below the text heading: gRNA transcript level of single U6-gRNA cassette using V3 scaffolds]

When we quantified the gRNA transcript levels produced by PX459.V3 the transcript levels were skyrocketing, even higher than dual U6-gRNA cassette. Note this is a, this V3 plasmid is a single U6-gRNA cassette, and this is Log2 scale. This is the V3 of the data compared to the other, the V2 and the dual guide cassette.

[Image changes to show a new slide showing two different graphs explaining the gRNA expression from single U6-gRNA cassettes below the text heading: gRNA expression from single U6-gRNA cassettes V3 lead to increased editing efficiency]

So, we used this V3 plasmid to test the less efficient gRNAs and the editing efficiency is now much better than before as you can see from the red bars, even higher than using dual cassette plasmid. This includes the gRNA that was less efficient in mouse ES cells. Now it has 100% editing efficiency which is what we expect when performing genome editing in mouse ES cells using our protocol. This suggests that a simple change in the gRNA’s coupled by replacing the T with C could help the gRNA expressions and lead to better editing efficiency. 

[Image changes to show a new slide showing a flow chart showing the progression of Hek293T through lipofection, harvest cells, extract pooled gDNA to NGS, and a graph appears below and a text heading: Compatibility of V3 scaffold for High-Fidelity Cas9: HF1 &, Lipofection without Puro selection]

We wondered whether this V3 scaffold is compatible with High-Fidelity Cas9, HF1-Cas9 and eSp1-Cas9, which both are engineered Cas9 with reduce off-target activity. So, we created constructs for HF1-Cas9 or eSp1-Cas9 with V3 scaffold and compared it with the normal scaffold. Note that this High-Fidelity Cas9 used the same gRNA scaffold as WT-Cas9.

So, we tested four different gRNAs to perform genome editing in Hek cells. In this experiment we didn’t perform puro selection because we also wanted to know whether V3 scaffold could give better editing efficiency in diluted situations. Diluted situations is more relevant for in vivo genome editing where we can’t do selection.

Transfection was done using Lipofectamine and here is the editing efficiency that’s on NGS. The left one here is the HF1-Cas9 data. On the right here is eSp1-Cas9. The blue bar is normal scaffold while the red bar is the V3 scaffold. Our conclusion here is that the V3 scaffold is compatible with High-Fidelity Cas9, and in some cases might give significantly higher editing efficiency in experiment without selection.

[Image changes to show a new slide showing a flow chart moving from Hek293T through nucleofection, puromycin selection, harvest cells, extract pooled gDNA to NGS below the text heading: Genome editing using SaCas9]

Our lab also used CRISPR system from staphylococcus aureus for genome editing, or SaCas9. SaCas9 recognised different PAM. Instead of recognising NEG PAM it recognised NAGRRP PAM. SaCas9 also has smaller size so it is an effective tool for in vivo genome editing with AAV delivery.

We have CRISP- SaCas9 plasmid which we call SaCas9-Puro-V2. This plasmid is similar to PX459-V2 except it’s express SaCas9 and Sa-gRNA. When we performed genome editing using SaCas9 we used the same protocol as before. We used nucleofection and puro selection. 

[Image shows two bar graphs appearing on the same slide showing editing efficiency data]

In this study we tested nine different editing using SaCas9 in Hek cells and here is the editing efficiency data. This is unlike the SpCas9 which consistently show near perfect editing efficiency, SaCas9 gives quite variable efficiency. 

[Image changes to show a new slide showing the SaCas9-Puro-V2 and SaCas9-Puro-V3 makeups and text heading appears: SaCas9 CRISPR scaffold also contains 4Ts and replacing T>C increased gRNA]

We noticed that Sa-gRNA scaffold also contained a string of four T’s and we wondered whether making the V3 version of it could give a higher cutting activity. So, we modified our SaCas9 plasmid to replace the T with C in the scaffold. 

[Image shows four graphs appearing on the same slide displaying data from the gRNA expression from U6 promoter]

We initially ensured that the V3 scaffold produced higher gRNA transcript level in Sa-gRNA and indeed it does. This is the gRNA levels comparing the different scaffold with the normal scaffold. Transcript level difference is huge. The right bars are the V3 and the left bars are the normal code and it is Log2 scale of change of the gRNA transcript.

[Image changes to show a new slide showing two bar graphs displaying data from Genome editing using SaCas9-Puro-V3]

We then performed genome editing using this SaCas9-puro with V3 scaffold and surprisingly we got much higher editing efficiency when using the V3 scaffold as shown with the red bar here, particularly from these three gRNAs which showed striking differences between the normal scaffold in blue versus V3 scaffold in red. 

[Image changes to show a new slide showing text: Summary, Genome editing using SpCas9 gives very high editing efficiency, Inefficient editing might be caused by inefficient transcription of gRNA, Using V3 gRNA scaffold is a simple way to ensure optimal transcription of gRNA, V3 scaffold is compatible for HiFi Cas9 (HF1 & eSp1), even gives better editing, Sa-gRNA V3 scaffold could improve gene editing using SaCas9 system]

To summarise this part we show that using SpCas9 we consistently get very high editing efficiency and if we get inefficient editing it might be caused by inefficient transcription of the gRNA. And using V3 gRNA scaffold is a simple way to ensure optimal transcription of the gRNA and the V3 scaffold is compatible with HiFi-Cas9, HF1 and eSP1, or even it could give a better editing efficiency. We also showed that Sa-gRNA V3 scaffold could improve gene editing when using SaCas9 system.

[Image changes to show a new slide showing various diagrams to show prime editing and text appears: Prime editing, Search-and-replace genome editing without double-strand breaks or donor DNA]

The next study I’m going to talk about is the newly invented prime editing, which has been shown to facilitate efficient precise editing such as making insertions or point mutations. We have done a bit of prime editing in the lab and we can tell you this technology is amazing. For some background, prime editing, is an engineered Cas9 developed by David Liu's lab in Harvard where they incorporate Cas9 nickase with reverse transcriptase. As a result performing HDR to create specific edit can use RNA template rather than the DNA template. What’s more interesting is that the RNA template donor, including the edit is attached in the same string of the gRNA, which is called pegRNA, or prime editing gRNA. 

[Image changes to show a new slide showing a flow chart explaining the mechanism of the Prime Editing PE3 system and text headings appears: Reverse Transcriptase, First nick RNA template binding, RNA template extension, Second nick, Resolution of non-edited strand]

Prime editing mechanism starts with nicking one of the DNA strands by the Cas9 nickase, and annealing of the pegRNA homology to the DNA. Then the reverse transcriptase extends the DNA by copying the RNA template that results in integration of the, of the edit. And they also found that additional nicking in the opposite strand improves the precise editing efficiency, a strategy they call PE3 system.

[Image changes to show a new slide showing two bar graphs comparing Precise Edits and Indels for Prime Editing PE3 and Standard HDR (ssOligo) under the text heading: Prime editing gives unprecedented precise editing efficiency]

David Liu and team observed high precise editing efficiency using prime editing. The blue bar here is the precise editing efficiency by prime editing which is really high. Usually you think Standard HDR method using oligo donor we get really low precise editing efficiency with high InDels as they are also showed here.

[Image changes to show a new slide showing a circular diagram showing the Prime Editing All-in-One construct with PEA at the centre of the circle and text linked by arrows: Cas9, Reverse transcriptase, Puro/GFP, 2nd nick gRNA, pegRNA]

So, we were interested in this prime editing technology and we would like to optimise this technique in the lab but we realised that the construct, the plasmid provided by David Lieu to perform prime editing wasn’t really handy. So, to perform prime editing using the PE3 system you need to contransfect three different plasmids, or four plasmids if you also want to do puromycin selections.

David Liu and team also didn’t perform selection in their experiment. So, we had a belief, we had a belief that prime editing efficiency must be better than what they observed. So, we thought let’s create a simple construct, an All-in-One construct where we can have all components in a single plasmid including selection marker so we can perform selections such as puromycin selection.

So, we generated this construct that we called PEA1 or Prime Editing All-in-One. This construct has the prime editor, the Cas9-nickase-reverse transcriptase with Puro or GFP selection marker and it has 2U6 from other cassettes for the expressions of the pegRNA as well as the 2nd nick-gRNA to perform PE3 system.

[Image shows red lines appearing on the diagram over the headings pegRNA and 2nd nick gRNA and text appears next to the red lines: Bbs1]

And to facilitate BG generation of prime editing construct of interest we designed this construct to have three Bbs1 golden gate sites so, we can easily change the gRNA, the editing template, and the 2nd gRNA. It’s so easy to create your personalised prime editing construct. 

[Image shows a pair of blue lines “Oligo pair 1 (gRNA)”, and  pair of red lines “Oligo pair 2 (repair template)” appearing on the left of the circle and two green  lines “Oligo pair 3 (2nd gRNA) appearing on the right of the circle]

You just need to order three pairs of oligos to carry your first gRNA, repair template and 2nd gRNA with specific overhangs. The overhangs for each oligo pair are different so, it’s unlikely you’ll get integrations of the insert in the wrong place.

[Image changes to show a new slide showing a diagram explaining the One step protocol (Digestion-Ligation) or DigLig and text appears moving down the screen joined by arrows: PEA1 plasmid, Oligo pair 1, Oligo pair 2, Oligo pair 3, Bbs1, T4 ligase, Cycle between 37C and 16C, Transform to competent cells, Pick colonies and prep plasmids, Verification (Bbs1 check digest and Sanger)] 

Once your ordered oligos coming you do this one step protocol that we call Digestion-Ligation or DigLig where you set up a single reaction to combine everything such as the PEA1 plasmid, all oligo pairs, the Bbs1 restriction enzyme, and the ligase. You put them in thermocycler, transform to competent cells, prep the plasmid, and verify the, verify the plasmid by Bbs1 digest and Sanger sequencing.

[Image changes to show a new slide showing a diagram showing the Bbs1 Diagnostic Digest and text heading and text appears: Easy generation of Personalized PE construct, 75% Triple Insert Integration Efficiency]

The chance in getting the correct prime editing plasmid is very good, 75% to 100% depending on your luck. This is the attempt from our Honours students in the lab with 75% success rate. This on Bbs1 Diagnostic Digest. The correct plasmid will stay circular after Bbs1 Digest.

[Image changes to show a new diagram showing the workflow chart of texting the prime editing starting with the Hek293T, HeLa, K562 and mES cells, and moving through Nucleofection PEA1-Puro, Puromycin selection, Harvest cells, Extract pooled gDNA, NGS, Analysis using PE-analyser from Rgenome]

When we test prime editing we use similar protocol for the standard CRISPR gene editing. We use nucleofections and puromycin selections. To see and quantify the editing we perform NGS and analyse the NGS using PE-analyser online tool. 

[Image changes to show a new slide showing a bar graph showing 24 edits in HEK cells from PEA1-Puro in Hek293T cells]

We tested prime editing using PEA1-Puro making 24 different edits in HEK cells. These edits are creating one bp and three bp insertions, one bp, and three bp deletions and making prime mutations in four different loci. These edits are identical to the experiments performed originally by David Liu in their paper. And this graph is showing the prime editing, or precise editing efficiency based on NGS. And we were so impressed with prime editing techniques because the efficiency to create the specific edits are really, really hard, something that was impossible before without prime editing. Nearly, nearly all of them gave more than 50% precise editing efficiency with one third of them gave more than 80% correct editing efficiency.

[Image shows a new graph appearing below of data from David Liu’s work on PE in Hek293T cells]

If we compared this with the efficiency from David Liu’s data using PEA, using our technique PEA1-puro and puro selection as we did boost the prime editing efficiency significantly. As you can see from those 24 different edits by David Liu, only two could give more than 50% efficiency indicating that our protocol using PEA1 could give higher prime editing efficiency.

[Image changes to show a new slide showing two graphs showing prime editing efficiency using PEA1-Puro in mouse ES cells]

We also tested prime editing using PEA1-Puro in mouse embryonic stem cells creating nine different edits in four different loci. We got quite variable efficiency but still high in general. In this experiment we used two different 2nd nick gRNAs and showed that different 2nd nick gRNA could make differences in prime editing efficiency. 

[Image changes to show a new slide showing two new graphs showing results from PEA1-Puro in HeLa and Pea1-Puro in K562]

Despite high editing efficiency in HEK cells it looks like prime editing is cell specific. David Liu found that prime editing is less efficient in other cell linings such as HeLa and K562. We also found the same thing even though we used the PEA1 construct and performed puro selection we still couldn’t get high prime editing efficiency particularly here in HeLa cells.

[Image changes to show a new slide showing text heading and text: Summary, Prime editing technology is amazing, PEA1 construct – Easy Prime editing experiments, PEA1 – selection – better efficiency, Prime editing need to be improved in other cells (HeLa)]

So in summary we agree that prime editing technology is amazing. We created PEA1 construct to make it easy to perform prime editing experiments. We showed that using PEA1 the prime editing efficiency is higher and another improvement we need to make for prime editing is ability in other cell lines such as HeLa cell or even primary cells that would be useful for therapeutic purpose.

[Image changes to show a new slide showing a photo of the Genome Editing Lab team smiling at the camera and text appears: Thanks to Genome editing lab, Paul Thomas, Sandie Piltz, Jayshen Arudkumar, Joshua Chey, Luke Gierus, Gelshan Godahewa, Stefka Tasheva, Ashleigh Geiger, Mel White, Caleb Lushington, Michaela Scherer, Mark Bunting, Athena Kang, V3 Scaffold, Joshua Chey, Jayshen Arudkumar, Luke Gierus, Louise Robertson, Prime Editing, Jayshen Arudkumar, Caleb Lushington, Joshua Chey, Gelshan Godahewa, Funding CSIRO SynBio fellowship, CSIRO, Denis Bauer, Kristie Jenkins]

Last but not least, I would like to thank Genome Editing Lab members, particularly Paul, the head of the lab, and as well as the people who performed the experiments. I also would like to thank CSIRO SynBio FSP for their funding. Thank you.

[Image changes to show Kristie talking on the main screen and the participant pane can be seen at the top of the screen]

Kristie Jenkins: Thank you. Anyone if you’ve got any questions make sure you post them in the Chat. You’ve inspired me, prime editing something that we’ve been thinking about doing and we haven’t actually got around to it yet. So, you’ve definitely inspired me to follow up on that so thank you for that. I’ve got a couple of questions here. One of the questions I got sent through was, “What do you see the real world applications of this type of platform technology?”. 

Fatwa Adikusuma: Sorry, say that again Kristie. 

[Image changes to show Fatwa listening on the main screen and then the image changes to show Kristie talking on the main screen and Fatwa listening in the participant pane]

Kristie Jenkins: What do you see as the real world applications for this platform technology?

Fatwa Adikusuma: The prime editing?

Kristie Jenkins: Yeah.

[Image changes to show Fatwa talking to the camera and then the image changes to show Kristie listening and Fatwa talking inset and then the image changes to show Fatwa talking on the main screen]

Fatwa Adikusuma: I, I think it’s great for our, in our lab we are interested in therapeutic purpose. So, I would say we would be able to use this technology to fix mutations in genetic diseases. For example cystic fibrosis, where majority got three nucleo pipe deletions at F508. So, we would like to be able to fix that mutations. So, I would see in the future it will be used for correcting deleted mutations.

[Image changes to show Kristie talking on the main screen and Fatwa can be seen listening inset in the participant pane at the top of the screen]

Kristie Jenkins: Amazing, thank you. Do we have any? I think we’re nearly out of time and I don’t have any other questions right now. So, if everyone would join me in thanking the speakers. If you want to turn your cameras off and show your applause or you can do your little clap gestures that would be fantastic. Thank you so much. Thanks both of you. I think we’ve covered off on all the questions so we’re good with that. And the next seminar will be on the 14th April and it will be Foundation Technologies and the invites for that will be circulated shortly. So, thanks. Thanks everyone. 

[Image shows Kristie waving on the main screen and continuing to talk and Fatwa can be seen inset in the participant pane waving at the camera]

See you next time. Bye.

[Image changes to show a K on the main screen and then the image changes to show Louise Burton’s photo on the main screen and Tim Doran can be seen listening in the participant pane]