Editing Genes Using Molecular Scissors

Friends,

In the last few years a revolutionary new technique has been developed to edit genes. In this VERITAS I will describe the technique and how it has the potential to completely transform the way we fight diseases and how it may help us come up with new disease resistant varieties of plants and animals.

The name of the technique is CRISPR-Cas9. It gives scientists a way to quickly and precisely cut a piece of DNA of any animal or plant and replace it with a different piece of DNA. Scientists have already done a lot of experiments with this technique and have achieved amazing results. Scientists in Japan have used this technique to create tomatoes that have a longer life by turning off genes that ripen them. Chinese scientists have created a variety of wheat that is extremely resistant to mildew. Scientists are proposing that people in haemophilia can be treated by editing their stem cells outside their body and then reinjecting these cells into the body. And as we will see in this article, scientists are trying to use this technique to conquer cancer, malaria and various other diseases.

A potential application of CRISPR-Cas9 is organ transplant. Every year millions of people around the world get into the waiting list to receive organ transplants. Most of them never get the organs and they die waiting. For many decades scientists have been proposing the idea of using animal organs to help people with organ transplant needs. The animal that has been identified is the pig and that is because the size of a pig’s organs is very close to that of humans. However, there is a problem: PERVs. The pig’s DNA is full of virus like structures called PERVs and these are able to infect humans. Till you solve the PERV problem, you cannot use pig organs in humans. Scientists at Harvard used CRISPR-Cas9 to edit the DNA in a pig cell to remove all 62  PERVs. And when these edited cells were mixed with human cells, none of the human cells got infected. Scientists are confident that in a few years people will be able to receive pig organs and not have to rely on human donors alone. This will solve the shortage of organs immediately and also put an end to the trafficking of human organs.

Now, lets look at how CRISPR works. The idea of genetic engineering is not new. More than 40 years ago scientists discovered how to take a piece of DNA of one organism and pasting it into the DNA of another organism to create a desired trait. This technique is called Recombinant DNA technology. But there lies a danger in this approach. One could also transfer viruses from  one species to another. Also, recombinant genes are not “natural” in the sense that in nature the two species may never have mated. For example, people with diabetes are treated with insulin that is produced by inserting human insulin gene into E. Coli or Yeast. So, it works but it is unnatural and this is one of the reasons for the huge public furore over genetically modified(GM) crops. CRISPR is different. DNA sequences from other organisms are not pasted into one organism’s genome. CRISPR is a technique to edit the existing DNA using “molecular scissors”.

The fundamental idea behind this revolutionary technique was not invented by us. It was discovered by observing bacteria and how they fight against invading viruses. In 1980s and 1990s scientists discovered that the DNA of bacteria contained short, repeating DNA sequences separated by short, non-repeating, “spacer” DNA sequences. These sequences were called CRISPR(Clustered regularly interspaced short palindromic repeats ). At first scientists did not understand the reason of these patterns. But then they found that these patterns are an essential components of the immune system of these bacteria. This is how it works: When a previously unknown virus invades a bacteria, a particular enzyme called Cas cuts a small piece of the invading virus’ DNA and inserts it as a spacer in the bacteria’s CRISPR DNA. So the spacer sections of a bacteria’a DNA are small pieces of the DNA of viruses that invaded it in the past. Thus the bacteria is keeping a memory of the genetic structure of viruses that have attacked it in the past. If in the future the bacteria is attacked by a virus of the same kind, the bacteria’s immune system can react very quickly. This is because it has a copy of a part of the virus’ DNA from the previous attack. The bacteria’s CRISPR system would now make two small strands of RNA with exactly the same sequence as the Virus’s saved DNA – these two RNA strands are called Crispr RNA ( crRNA). A Cas9 molecule attaches to the CrRNA and together this system goes to attack the virus. The CrRNA is used to find the exact location of the virus’s DNA where the attack will happen. When the match between the CrRNA and the virus’s DNA occurs, the Cas9 cuts the virus’s DNA rendering its gene ineffective and making the virus unable to continue the attack. So, the CrRNA acts as a guide to where the cutting will take place and the Cas9 acts as a molecular knife which does the cutting. This is shown in the following image from Wikipedia.

Crispr

Scientists thought of using this technique to edit human, animal and plant DNA for our benefit. We can create a RNA sequence of any base pairs. The first step is to create a RNA sequence of the DNA sequence that we are targeting. They called this guideRNA. If we combine this with a Cas9 molecule, we have a very precise DNA cutting tool. The guideRNA will go to the right place in the target organism’s DNA and then Cas9 will cut it off. So we can remove genes, or even modify them. Once we cut one sequence of the DNA, we can also replace it with a similar sequence with slight modifications. For this to happen, scientists also send a repair template. When the target DNA is cut, it tries to repair the cut. A DNA strand typically repairs the cut based on the sequence of the complementary strand. But if we use CRISPR-Cas9 to cut both DNA strands, then the DNA has no way of knowing what sequence to recover while repairing. If we do nothing, the DNA will simply bind the two cut portions together. But if we send a repair template, the DNA will think that it is the complementary DNA strand and creates a sequence just like it. So, it “looks” at the repair template and creates a sequence just like that. So we fool the target DNA to repair its cut portion but instead it making the same sequence as the original, we create a slightly different sequence- one that is beneficial to us. This technique of fooling the DNA to create a new sequence based on the template we send it is called homology directed repair.

Thus we see that we can use the CRISPR-Cas9 mechanism to either remove some harmful genes by cutting them off both strands of the DNA or we can modify the genes by cutting off the original gene and then fooling the DNA to repair it using the template that we give it. Thus using this technique we can achieve very precise gene editing results. And it can be done at a small fraction of the cost and time compared to traditional genetic engineering methods. It is estimated that CRISPR techniques cost about one hundredth of the cost of traditional genetic engineering methods. Also, the time taken to do one CRISPR experiment is about a week whereas traditional genetic engineering methods could take more than a year. And to top it all, CRISPR is a very precise technique- something that traditional techniques cannot even hope to match. So, we have a revolutionary new scientific tool in our hands. And the possibilities are endless.

Scientists have tried to use CRISPR to cure mice of HIV. And they have been successful. Scientists were able to cure live mice of HIV infection using this technique. Scientists also believe that CRISPR techniques will help us battle and possibly cure cancer. If we can use CRISPR to modify the DNA of the cells of the immune system of our bodies and command these cells to seek out and destroy the cancer cells, we will win the battle over this dreaded disease.

There is another way in which scientists are thinking of using CRISPR-Cas9 system: Fighting mosquito borne diseases like Malaria, Dengue and Zika. The mosquito has been the cause of billions of deaths in human history. Mosquito borne diseases have caused more deaths than all wars in history combined together. Every year nearly half a million people die of malaria. But mosquiotoes do not themselves cause malaria or any other disease that we associate with mosquitoes. Mosquitoes are carriers of the microbes that cause these diseases. So how about using CRISPR-Cas9 to change the DNA of mosquitoes to be resistant to these mosquitoes. If we do that, mosquitoes will not carry the microbes and will not spread diseases. But there is a problem: how many mosquitoes will scientists modify? If they modify just a few, they will not be able to make a big difference. The aim should be to create a DNA change that is carried by the offspring also. And the DNA change has to be dominant so that when a genetically modified mosquito mates with a wild one, the changed DNA is carried on to the next generation. So the gene modifications that we do should be dominant over the original gene. Scientists have devised such a mechanism- again using CRISPR-Cas9.  It is called gene drives. We have already created genetically modified mosquitoes that are not capable of spreading malaria and with gene modifications that will always be dominant. If we use this technique the hope is that within a few generations most mosquitoes will be resistant to the microbes carrying deadly diseases.

So, we have a very powerful new tool in our hands. But we also need to be very careful. Whenever in history humans have had access to a powerful tool, we have also misused it and caused immense harm to ourselves. With CRISPR we need to be careful not to use it to satisfy the lust for power or wealth. For instance, a rogue country may use it to create an army of super-humans. Or someone could use it to create new kinds of diseases and use those during wars. We have a great responsibility to use this power carefully. The aim of science should be to elevate the human race and not to satisfy our lowest animal desires and needs.

 

Kanwar

 

 

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Go wondrous creature, mount where science guides

go measure earth, weigh air, state the tides,

instruct the planets in what orbs to run

correct old time, regulate the sun

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