Gene Editing and CRISPR Made Simple

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CRISPR is a powerful gene-editing technique that is transforming the field of genetics. Faster, easier and cheaper than previous gene modification methods, the dream of treating cancer or curing hereditary gene diseases is one step closure. However, with the power to change DNA come ethical implications. CRISPR gives us the ability to alter the evolution of an entire species. Scientists are excited but are aware that they need to be careful how they use this revolutionary gene editing tool.

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What if we could use one tool to bring species back from extinction? Wipe out malaria? Cure genetic disease? Make food allergen-free? These are all ideas scientists believe they can realize using a new gene-editing technique called CRISPR-Cas9.

Professor Jennifer Doudna
It stretches from human therapeutics to agricultural applications to thinking about, “How do we make better biofuels?”

Prof David Tscharke
It’s massive. It allows a vast amount of biology to be done that we just couldn’t do before.

Dr Sara Howden
CRISPR enables us to very precisely go in and find the gene that is dysfunctional and genetically repair it.

Prof George Church
It’s Darwin on steroids.

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What is CRISPR? How does it work? And how might it change our DNA, and our world? All organisms have a genome, which is a string of DNA that can be billions of letters long. The DNA code is made up of four base pairs – A, T, G and C – the source code of life. DNA divides, replicates and recombines, making us who we are. A small change in the code can result in a disease forming or a physical trait changing. So, why would we want to edit our genes at all? It’s estimated there are over 10,000 single-gene disorders, and currently only around 5% can be treated. By editing DNA, we can not only eliminate the chance of disease, but also change how a person develops…and fundamentally understand what a single gene does.

Dr Mark Tizard
For many years, we had this situation of, “OK, we can sequence things. Now what do we do with it?” CRISPR has given us that tool to be able to go in and say, “We’ve got the genome sequence. Now we can understand what that genome sequence means and be able to change it in beneficial ways.”

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So what is CRISPR?

Prof David Tscharke
CRISPR stands for ‘clustered regularly interspaced short palindromic repeats’, which doesn’t really tell you what it does at all.

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Don’t let its full name put you off. CRISPR quite simply is the perfect machine for chopping up DNA. And its discovery was somewhat an accident.

Dr Mark Tizard
These repeat sequences kept cropping up and people didn’t know what they were, and they were this big mystery for decades. They were looked at like an oddment, an artefact, but people went after it and said, “No, this has got to mean something.” And then to uncover that this was in fact a viral immunity system in bacteria – fantastic piece of basic research. And then someone has the talent to recognise that it could be adapted from bacteria and put across into eukaryotes, mammals, birds, whatever. And, you know, it’s just been a sea change in research for us all.

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The molecular machine is made up of two molecules – a protein called Cas9 that cuts DNA like a pair of genetic scissors, along with a piece of guide RNA that tells the Cas9 exactly where to cut the DNA. Professor Jennifer Doudna is one of the original researchers who realised the potential of CRISPR.

Professor Jennifer Doudna
We like to talk about science in our household. My husband’s a scientist and I was telling my son about this molecule, about Cas9, and I was telling him that, you know, it’s like a little machine that goes into the cell and it floats around the DNA until it finds a site that matches its RNA, and then it makes a cut, you know, and then it falls off and then the cell can repair the cut. And he said, “Well, so what? Why do we care?” And I said, “Because we can control where it’s making that cut, we can actually tell cells to fix DNA at a particular place and make a little change in the DNA code of the cell.” And so he looks at me and he says, “Mom, you mean you could change my DNA?”

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Genetic modification has been around for some time, so how does CRISPR differ? Harvard professor George Church is revolutionising the use of this technology. He’s trying to bring back the woolly mammoth, and holds the world record for the most number of genes edited using CRISPR.

Prof George Church
It is a form of genetic modification where you not only can add things, which is what happens in transgenics, where you’ll move a gene from one organism to another, but here you can also remove or precisely edit things. And when editing, you cut the DNA in a precise location which is determined by about 20 A, Cs, Gs and Ts that you choose. And when you cut it, then you can repair it in either a precise or imprecise way.

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That’s the real power of this tool. It’s opening up a whole new research area, and any molecular biologist can do it.

Prof Jennifer Doudna
I like to call it democratising technology. It means that laboratories don’t have to be particularly skilled in protein engineering, they don’t have to have a lot of money to spend on this technology, because it’s relatively inexpensive to use, and scientists don’t have to have any special training to do this. So it just opens up a technology to many more labs than was possible in the past.

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One of the most exciting prospects is using this molecular machine as a therapeutic tool to treat genetic disease. The closest researchers have come to editing human genes to treat disease is in tiny organs made from stem cells that are grown in a Petri dish. These are known as ‘organoids’.

Prof George Church
You can make something that’s very similar to a kidney, intestine, liver or brain structure, then you can test whether the change that you made with CRISPR has a particular effect on the health of that tissue.

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In Australia, Dr Sara Howden and Professor Melissa Little are leading the way in understanding kidney disease by creating CRISPR-edited kidney organoids.

Prof Melissa Little
We use gene editing to actually fix the mutation that we think is causing the disease.

Dr Sara Howden
We go ahead and take a skin biopsy from these patients, we turn them into stem cells and at the same time we correct that mutation that we think might be causing their disease. Then we use those cells to make little mini kidneys in a dish – what we call ‘kidney organoids’ – to try and understand how those diseases might be leading to kidney dysfunction. So, what we have here is skin cells. These were actually taken from a newborn baby who had been circumcised. So I’ve added my skin cells to a tube containing the factors that will turn these cells into stem cells, and also the gene-editing factors, which includes the CRISPR-Cas9.

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Sara applies an electric pulse that pokes transitory holes in the cell membrane, allowing the CRISPR-Cas9 to get inside the cell.

Dr Sara Howden
Basically, the cells do the rest.

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These stem cells become kidney cells, which Melissa uses to make mini kidneys in a dish.

Prof Melissa Little
This is actually an organoid. We grow the cells in a culture for about seven days and then we put them onto this filter. So, we can see the cells are starting to form little tubular structures, and you can see these with the naked eye.

Prof George Church
So it allows you to do something you couldn’t do on people, you can do on the cells or the organs of people.

Prof Melissa Little
We can look at a little organ from the patient and a little organ from the patient that is only different because we’ve gene edited to correct that mutation. So, it’s the best way we can model a disease.

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By making edited organoids, medical researchers have been able to revolutionise their understanding of gene function in disease, and in the future we may be able to see CRISPR being used as gene therapy.

Dr Sara Howden
We need to make sure they’re safe, need to make sure they’re effective over the currently available treatments. But I think many of us believe it’s only a matter of time before we start to see these types of therapies in the clinic.

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But people are generally concerned when scientists talk about genetic modification. Professor Rachel Ankeny examines ethical and policy issues related to gene editing.

Prof Rachel Ankeny
In the biomedical domain, where this has the potential to cure very serious diseases from which people suffer, the public is much more likely to be open to allowing investigations in that area. But as soon as it goes offshore or underground or it’s not visible, for whatever reason, I think that’s where the hesitance really creeps in, and correctly so.

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Branching out from therapeutics, CRISPR is having enormous impact on fundamental medical research, spanning from cold sores to cancer. Professor David Sharkey’s lab is trying to understand the basic biology of the common cold sore virus.

Prof David Tscharke
Trying to understand why the virus stays dormant, and importantly also why it comes out of that state and can cause what appears to be a new infection is a really important problem in virology.

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David uses CRISPR to engineer herpes viruses, then introduces a protein that glows green when the gene he is studying becomes active. This allows him to uncover whether the virus truly becomes dormant.

Prof David Tscharke
The virus will just sit there doing very, very little, but what we’ve discovered, it’s doing a little bit more than nothing.

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It was commonly thought that the cold sore virus lay dormant in a ganglion at the base of your brain. With the help of his CRISPR-edited glowing viruses, David has found that the virus is always active and that the human immune system is constantly fighting it.

Prof David Tscharke
Trying to understand when the body loses control we think is going to help us understand why some people, for example, will have to suffer very frequent cold sores, while the vast majority of people never see the virus again.

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Our understanding of the genes switched on in a cancer cell is also leaping forward, thanks to CRISPR.

Dr Marco Herold
So, what’s very exciting about CRISPR for us is that it’s the first time you can manipulate the genome in such a fast and easy way.

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Dr Marco Herold modifies a virus to contain the guide RNA and Cas9. This is how the molecular machine gets into the cancer cell.

Dr Marco Herold
And this virus contains the enzyme Cas9 and also the guide molecule to introduce a CRISPR.

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Once the modified virus infects the cancer cell lines, it can quickly knock out all 20,000 genes in the genome, one at a time.

Dr Marco Herold
We have one of these genes which we have shown genetically in preclinical models if you take it away, then it kills them.

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By removing this gene in a test tube, the cancer cell stops growing.

Dr Marco Herold
And now we are going down the path and developing drugs in order to target this protein.

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It’s early days, but this could lead to a new way to fight cancer.

Dr Marco Herold
Not a be-all, end-all, but at least a novel treatment access for not only one type of cancer but for many different cancers.

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Traditional gene editing has always been most hotly contested when it’s applied to our food. Doctors Tim Doran and Mark Tizard are leading Australia’s efforts in using CRISPR for agricultural applications. Theirs is a long-term partnership.

Dr Mark Tizard
22 years.

Dr Tim Doran
Yeah. We started working together in the UK. I was in London doing a post-doc and Mark was already established in the labs.

Dr Mark Tizard
We’re interested in a range of genes that are involved in both health and response to disease, allergens that occur in eggs and also issues to do with sex selection in chickens.

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The egg industry currently culls half of the breeding chicken population, as males don’t lay eggs. How can CRISPR help?

Dr Mark Tizard
We’re trying to introduce a technology that will allow industry to identify those embryos before they even grow into a chick. When the egg is laid, there’s a developing embryo inside, and we can access that and make modifications to those germ cells, which will then appear in the following generation.

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They tag the sex chromosomes with a green flag so the modified male embryos fluoresce green.

Dr Mark Tizard
So that little ball of cells will be fluorescently green, we can see that through the shell and we can take the egg out and chill it and stop it from growing.

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Only the males carry the green protein. The females are not genetically modified. Time will tell if the regulators and the public jump on board to use gene editing in this way.

Dr Mark Tizard
We’ve done a lot of studies asking the general public about their views on, say, genetic modification of crops, and they’re still not sure what’s in it for them. And at the end of the day, although, you know, we’re a community, we look outward, we want things, you know, to be good more generally, we’re very concerned about “What are the effects of these things going to be on ourselves and our families?”

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Tim does have a personal reason for modifying food. His daughter has food allergies.

Dr Tim Doran
I have an 11-year-old daughter who is allergic to eggs.

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So Tim and Mark have also put their efforts into making allergen-free eggs.

Dr Tim Doran
There are four proteins within egg white that cause allergy. The way we use CRISPR in this application is as an editing tool, so we’re essentially rewriting those regions of the gene that are recognised by the immune system and cause an allergic reaction.

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Allergen-free eggs could be used in vaccines as well as food products.

Dr Tim Doran
So it will need to go through the regulatory process, but from my perspective, I’ve found that parents that have children that have food allergies are very used to understanding what’s in different food types, and I think they’ll go to the effort of really trying to understand the science behind what we’re trying to achieve. Show me a kid that doesn’t want to have a birthday cake on their birthday. That’d be the goal – a nice big birthday cake that she could have that tastes pretty good.

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This technology has the potential to change our DNA and the DNA of all organisms alive and extinct.

Dr Mark Tizard
Everything’s going to change now, and it has. And there are more changes to come, I believe.

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It’s a powerful tool that needs to be used wisely.

Prof Jennifer Doudna
I’m really involved right now in a lot of ethical discussions around the use of the CRISPR technology, how we can be responsible about using it, how we can make sure that we’re explaining to the public what this technology really is – because, you know, the science is going 1,000 miles an hour. I think that it’s a very, very exciting technology that’s going to do a lot of good in human society and for human health.

STORY CONTACTS

Dr Mark Tizard
Molecular Biologist,
AAHL, CSIRO

Prof Jennifer Doudna
Molecular Biologist,
University of California – Berkeley

Prof George Church
Geneticist, Harvard University

Prof Melissa Little
Molecular Biologist,
Murdoch Children’s Research Institute

Dr Sara Howden
Molecular Geneticist,
Murdoch Children’s Research Institute

Prof Rachel Ankeny
Bioethicist,
University of Adelaide

Prof David Tscharke
Virologist, JCSMR,
ANU (Australian National University)

Dr Marco Herold
Molecular Geneticist,
Walter and Eliza Hall Institute of Medical Research

Dr Mark Tizard
Molecular Biologist, AAHL,
CSIRO

Dr Tim Doran
Molecular Biologist, AAHL,
CSIRO

Article Credit: ABC