What is CRISPR-Cas9? Revolutionary gene editing technology explains



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Until recently if you wanted to make, say, drought-resistant corn plants, your choices were very limited. You can choose selective breeding, try bombarding seeds with radiation in the hope of encouraging beneficial changes, or choosing to insert pieces of DNA from other organisms completely.

But this approach is long-winded, inappropriate or expensive – and sometimes all three at the same time. Enter CRISPR. Right and cheap to produce, these small molecules can be programmed to edit organisms' DNA to certain genes.

Cheap and relatively easy development of gene editing has opened up many new scientific possibilities. In the US, the edited longevity mushroom CRISPR has been approved by the authorities, while elsewhere researchers are playing with the idea of ​​creating spicy tomatoes and peach-flavored strawberries.

But game changer technology can have the greatest impact on human health. If we can edit problematic mutations that cause genetic diseases – like hemophilia and sickle cell anemia – we can end it all. However, the road to editing human genes is full of controversy and a harsh ethical dilemma, when the news at the end of 2018 that – against all ethical guidelines – a Chinese scientist secretly created the first gene-edited baby.

This is all you need to know about the complicated and sometimes controversial technology that drives the revolution in editing genes.

What is CRISPR?

CRISPR evolved as a way for several species of bacteria to defend themselves against virus invaders. Every time they encounter a new virus, the bacteria will capture DNA fragments from the viral genome and make copies to be stored in their own DNA. "They collected a series of sequences they had faced," said Malcolm White, a biologist at the University of St Andrews, "this [bacteria] basically carrying a small library in their genome. "

To stick to the analogy of the library, pieces of DNA from this virus are like small books – each containing data that allows bacteria to recognize and quickly kill viruses the next time they are attacked. And among these useful pieces of DNA there are repetitive pieces of DNA that are a little less useful which make them separate – like a molecular bookend.

It is this recurring DNA segment that gives the CRISPR its name – Clustered Regular Interspaced Short Palindromic Repeat – but it's really a piece between these repetitions which makes CRISPR very useful. These useful bits, rather unhelpful, are called spacers, and each contains a reference to the DNA of a virus that bacteria (or their ancestors) have encountered in the past. When a previously invisible virus attacks bacteria, it adds another spacer to the previous attack library.

When a virus from the same species attacks again, the spacer that matches the viral genome takes action. This is somewhat similar to how our own immune system can recognize a flu virus if we have got a flu vaccine that year. The sequence of spacers is converted to RNA – a molecule that contains a message from DNA – and hunts for the appropriate DNA part of the virus. Once found, the enzyme attached to the RNA rope acts as a pair of biological scissors, cutting off the target DNA and making the virus harmless.

You may have heard of this system referred to as CRISPR-Cas9 and also ordinary CRISPR. In this case, the Cas9 bit refers to the enzyme used to cut the target DNA. "We can program [Cas9] it's very easy to target one DNA sequence and be very specific so it won't cut anything that is even the same order, "White said. There may be other types of enzymes involved in gene editing – for example Cas12 and Cpf1 – but all work in the same basic way.

How does it work?

Of course, all of this is only useful if you are a bacterium. So how do we turn the anti-virus defense mechanism into something that can enable us to edit the human genome as we please?

Instead of relying on bacteria to make molecules for them, scientists have worked on how to make their own versions of CRISPR molecules in the laboratory. To start, they need to work on the part of the DNA they want to target. For sickle cell anemia conditions, which are caused by errors in a single gene, this is relatively easy, because we have sorted the genes that cause this disease and know the exact genetic code we are trying to target.

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Before we talk about opening zippers and cutting DNA, it's good to learn the basics of how DNA is structured. Holding together a known DNA double helix is ​​four different nitrogen bases: adenine (A), thymine (T), guanine (G) and cytosine (C). The order of these bases determines everything about us, which speaks genetically. Eye color, how high we are, whether we are susceptible to certain diseases, everything is written in base pairs in our genetic code.

Like teeth on zippers, these bases always pair with their complementary bases. Couples always pair with T while G always pairs with C, repeatedly until you reach three billion base pairs that make up the human genome.

But DNA is not widely used to remain locked in a double helix – it is necessary to get that information out there and into the cell where it can be used to make protein, which is the building block of almost everything in our body. To do this, the DNA unzips itself, breaking the base pair until they flap inside the cell.

The base pair that flaps and doesn't pair in the meantime matches the short RNA segment that contains their own base. RNA shares three bases with DNA – G, C and A – but T is always replaced by U (urasil). Similar basic pairing rules apply, so that the base of the exposed DNA G will pair on an RNA C basis while the base DNA A will pair with U. If you have an open DNA sequence from GAC, for example, you will end up with an RNA sequence from CUG.

Scientists use these basic principles to make their own CRISPR molecules which, as we pointed out above, are short stretches of RNA. All you need to do is open a DNA sequence that looks interesting – like bits containing mutations that lead to sickle cell anemia – and build complementary RNA sequences, with attached DNA cutting enzymes. This is like starting with one side of the zipper and using it to build a side zipper that fits but opposite that fits inside.

After you get the CRISPR molecule, you need it to get your target cell. Fortunately, the virus is no more than injecting goods into other cells, so bringing up CRISPR molecules into benign viruses is one very useful way to incorporate CRISPR into cells that have been used in many studies involving mice.

Now CRISPR-Cas9 can really work. The Cas9 enzyme starts with opening the zipper from the DNA double helix while the RNA molecules sniff along as long as the open base pair is looking for the perfect pair. After the perfect pair is found, Cas9 cuts the troublesome gene before repairing the remaining DNA part. Other enzymes can add inserted genes rather than delete them, but the basic process of unzipping, recognizing and editing remains the same in various CRISPR molecules.

What is CRISPR used for?

CRISPR is very attractive to the agricultural industry, which is always looking for ways to engineer disease-resistant and weather crops that will increase their yields and, subsequently, their profit margins. In October 2015, plant biologists at Pennsylvania State University in the US gave US Department of Agriculture (USDA) regulators with edited mushrooms so that they became brown much slower than normal mushrooms.

A year later, the USDA confirmed that the same fungus would be cultivated and sold without having to go through the institution's regulatory process for genetically modified foods. Now, non-brown mushrooms are not the most thrilling food, indeed, but this USDA is a considerable problem because it implies that CRISPR-edited plants may be able to avoid some environmental attacks aimed at GMO plants.

And it's not just mushrooms that get love for CRISPR. In Australia, a scientist has used CRISPR to make bananas immune to deadly fungi that threaten to destroy the world's fruit plants, while others work using technology to make natural decaffeinated coffee or finally engineer perfect tomatoes.

Timeline: When was the CRISPR found?


2005
After characterizing CRISPR in 1993, Francisco Mojica at the University of Alicante in Spain became the first to hypothesize that DNA sequences are part of a bacterial adaptive immune system.
2007
Scientists at Danisco, a Danish food research company, proved experimentally that CRISPR was part of the bacterial immune system and that Cas9 deactivated the invading virus.
2011
The Emmanuelle Charpentier group at Umea University in Sweden showed the role of tracerRNA in guiding Cas9 to its cellular targets.
2012
Emmanuelle Charpentier and Jennifer Doudna at the University of California, Berkeley simplifies the CRISPR system by combining together various elements into one, synthetic guide

Even though the world of agriculture provides some examples of CRISPR farthest along the action, the stakes are much higher when it comes to human health. Animal studies have been carried out to use CRISPR to treat sickle cell anemia and hemophilia – two promising candidates for the treatment of CRISPR because they are determined by a small number of mutations. In the case of sickle cell anemia, this condition is caused by only a single base pair mutation in one gene.

The more genes involved in a condition, the more difficult it is to use CRISPR as a potential solution. "There aren't many human diseases where only one gene mutates," White said. Certain cancers, for example, are associated with many mutations in different genes, and often the relationship between genetic mutations and cancer risk is poorly understood so there is no guarantee that – even if we can use CRISPR to correct the wrong genes – that's all & # 39; d to be all kinds of panacea for cancer.

Why is CRISPR controversial?

Late last year, He Jiankui, a researcher at the South University of Science and Technology in Shenzhen, shocked the scientific world when he claimed responsibility for the first human edited CRISPR in the world. He reportedly took an embryo from a partner whose father was HIV-positive and an HIV-negative mother and used CRISPR to edit genes that control the protein channels that HIV uses to enter cells.

Experiments – detailed in YouTube videos, not peer reviewed journals – are widely condemned by scientists. "It is widely recognized that science is not ready for clinical applications," said Sarah Chan, bioethics expert and director of the Institute of Medicine, Life Sciences, and Law Law at the University of Edinburgh at the time. "Much needs to be done to resolve uncertainty, and to try and understand the risks."

Even though He's study completely violated ethical boundaries, it caused one of the great ethical puzzles when it came to CRISPR. The problem is it's not easy to use CRISPR to change your genome once you grow up – you have to find a way to introduce molecules to each target cell.

This may be achieved for conditions such as sickle cell anemia, where you only need to change DNA in red blood cells. By using CRISPR to edit bone marrow – where red blood cells are produced – you might be able to target the percentage of cells that are relatively small and still improve their condition.

But if you want to change a person's entire genome, you need to edit their DNA when they are a little more than a small group of cells. This leads to all kinds of ethical problems. Why stop identifying and destroying genetic diseases, for example, if we can also change embryonic DNA so that the babies produced are more likely to be intelligent, or handsome?

"What if we want to change the future life span, or intelligence, or the potential for Alzheimer's disease or whether they become bald when they reach middle age," White said. "People must agree to what we want – it's not up to scientists."

Although editing of human genes raises some of the biggest ethical questions, many things are not very clear in terms of agriculture. In July 1018, the European Court ruled that the future of gene-edited plants became doubtful when it confirmed that CRISPR-edited plants would not be exempt from existing regulations limiting the planting and sale of genetically modified plants.

Genetically modified plants – usually by inserting genes from one organism to another – have long been ruled out in Europe, despite their popularity in other parts of the world. Regardless of the scientific consensus that GM food is safe for consumption, the main editorial warning is franken food & # 39; and lobbying from environmental groups helps keep GM plants away from human consumption.

But agricultural advocates for CRISPR hope that new gene editing technology will provide an opportunity to improve this balance. The ECJ decision means that any CRISPR edited food that will be planted or sold in the EU must pass a strict security test that unedited plants (or plants made using certain techniques such as radiation mutations) do not have to be faced. For now, at least, one of the biggest obstacles facing CRISPR is not science, but public relations.

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