Magic lamp for garden repair / Margaret Knox

A method for editing DNA based on bacterial "memories" could revolutionize medicine. But some are worried that the method could get out of control

The era of genetic engineering began in the 70s when Paul Berg spliced ​​DNA from a bacterial virus into a monkey virus, and Herbert W. Boyer and Stanley N. Cohen created creatures whose genes inserted into their genomes remained active for generations. In the late 70s, Genentech, Boyer's company, produced insulin for diabetics that it produced from a transgenic E.coli bacterium that contained a synthetic gene from a human. At that time, scientists in the US began using transgenic mice to study diseases.

These victories changed the face of medicine. But they had two main limitations: the tools available to the scientists were imprecise and unsuitable for a large scale. In the 90s, researchers overcame the inaccuracy, when they designed enzymes that could cut specific places in DNA: a huge improvement compared to inserting DNA into cells at random, hoping that desired mutations would be obtained. But they still had to "tailor" a new enzyme to each desired target of a DNA sequence, a slow process that requires great meticulousness.

Then, two years ago, in 2013, a small group of researchers working in the laboratories of Emmanuel Charpentier at Umeå University in Sweden, and Jennifer Dudna of the University of California, Berkeley, reported the discovery of a genetic mechanism in cells that allows scientists to edit genomes with unprecedented ease and speed. Immediately afterwards, teams of scientists at Harvard University and the Massachusetts Institute of Technology (MIT) showed that with this method it is possible to create many changes in the cell's genome at the same time and with great precision.
Advances have already accelerated the genetic modification industry in ways that are likely to have profound and beneficial effects in the fields of genetics and medicine. Scientists can now engineer transgenic lab animals at will in weeks and save about a year of work. Researchers use the method to find a cure for various diseases such as HIV, Alzheimer's and schizophrenia. However, the method has made the genetic modifications so easy and cheap that some ethics experts foresee possible negative results.
This method is called "CRISPR" (clustered regularly interspaced short palindromic repeats), named after conserved DNA sequences, which allow bacteria to remember viruses that attacked them. Scientists have been studying these strange sequences since they were discovered by Japanese researchers in the late 80s. However, the ability inherent in CRISPR sequences to serve as a tool for gene editing only became clear when Doudna and Charpentier's teams figured out how to use a protein that acts as an enzyme, called Cas9.

The power of RNA
Doudna and Charpentier met in 2013 at a scientific conference in San Juan, Puerto Rico. They had a lot in common: they both headed research groups that studied how bacteria defend themselves against viruses. Both conducted studies that proved that bacteria recognize viruses that attack again with the help of "memories" left by the previous invader in the bacterial DNA.
Shortly after their meeting, Dudna and Charpentier decided to join forces. Charpentier's laboratory in Umeau collected hints that streptococcus bacteria use a single enzyme, Cas9, as a kind of sword with which they cut down viruses that have breached their cell wall. Dudna's lab at Berkeley took on the task of understanding how Cas9 works.
By a strange coincidence, like the ones that underlie many scientific discoveries, it turned out that two researchers, Krzysztof Hilinski in Charpentier's group and Martin Yeink, then in Dudna's lab, grew up in neighboring cities and spoke the same Polish dialect. "They started talking to each other on Skype, a 'chemistry' was created between them and they started sharing data with each other and coming up with ideas for experiments," Dudna said. "From there the project took off."
Scientists in both labs realized that Cas9 could be used in genome editing, a type of genetic engineering that uses enzymes as molecular cutoff numbers. The enzymes, called nucleases, break the double-stranded DNA at certain sites, and the cell, which repairs the place of the break, sometimes incorporates new genetic material that the scientist places in the nucleus. When Dudna and Charpentier began to collaborate, the most widely accepted method available to neutralize or change a gene was to adapt to each case the enzyme that would be able to find and cut the target sequence in the DNA. In other words, the scientists had to sew for each genetic modification a new enzyme that would target the desired DNA sequence.

Doudna and Charpentier realized that Cas9, the enzyme that the streptococcus bacterium uses for immune defense, uses RNA as a guide that leads it to its target in the DNA. Looking for the target, the attached Cas9-RNA runs along the DNA, seemingly at random, until it finds a promising target. It turned out that detection is the way the Cas9 enzyme searches for that short sequence of DNA. The Cas9 enzyme sticks to this sequence, opens the double helix and checks if the strands in it match the guide RNA. Only when the DNA matches the guide RNA, Cas9 will cut it. If it is possible to harness this system guided by RNA, researchers will not have to create a new enzyme every time to reach every goal in the genome. Editing will be simpler, cheaper and more efficient.

After months of collaborative research on Cas9, the transatlantic team had a breakthrough. Dudna remembers the moment clearly: Yanek, then a postdoctoral fellow, was running Cas9 experiments in a lab overlooking the Greek Theater on a wooded hillside at the edge of Berkeley's campus. One day, he came to Dudna's office to discuss the results and they talked about something that came up in the collaboration with Hilinsky: in nature, Cas9 in the streptococcus bacterium uses not one guide RNA, but two to tune to the right spot in the double helix of the invader's DNA . What will happen if they artificially connect the two guides to one guide without harming their effectiveness? The need to change one sequence instead of two will speed up the work of genetic engineering to the greatest extent. It is much easier to build a guide RNA than to build the mediators in the binding of the respective stitched enzymes, in the old method, on their complex coding charts.
"It was one of those moments when you look at the data and suddenly a light bulb goes on," Dudna says. "We realized that we could build these RNA molecules as a single guide. A single enzyme and a single guide will be a powerful tool. I felt chills run down my spine, 'My God, I'm running to the lab, don't go.' If it works…”
And it worked, with consequences that her cousin, with all her enthusiasm, could not even imagine. When Dudna and Charpentier published the results of their research on CRISPR-Cas9 on August 17, 2012, scientists in the field immediately recognized the potential inherent in the research results and a global race began to test the applications.
The race for commercialization
In 2013, the researchers already adapted the CRISPR-Cas9 system to work in plant cells and animals much more complex than bacteria, and even contemplated fictional applications such as bringing back to life Neanderthal man and woolly mammoths. A team led by geneticist George Church at Harvard University used CRISPR-Cas9 to modify genes in human cells and opened up a whole new world of healing possibilities.
Not surprisingly, money started flowing into CRISPR-Cas9 work. A little over a year ago, Dudna, Church, Peng Zheng and other researchers teamed up to launch the company Editas Medicine with venture capital of 48 million dollars in order to develop a new type of medicine based on CRISPR. (The company has not yet announced which disease it will focus on first.) In April 2014, CRISPR Therapeutics was launched for the same purpose with an investment of $25 million. Treatments developed by such companies are still a long way off. But companies that supply research products to laboratories are already shipping ready-to-inject CRISPR to customers around the world, and mice, rats and rabbits that have been modified in this way.
On a steamy day in the summer of 2014, I visited SAGE Labs in St. Louis, the first company to acquire the rights to Dudna's CRISPR method for the genetic engineering of rodents, so I could see for myself how the method works. Sage supplies goods to about 20 pharmaceutical giants, many universities and biotechnological institutes. (In September 2014, Sage was bought for $48 million by Horizon Discovery Group, a biotech company founded in Cambridge, England, which was already producing its own Crispr.) At Sage, located in several low-rise office buildings in an industrial area, scientists take orders online. For example, an order from Sacramento, California, of 20 rats in which the Pink1 gene is damaged, for the purpose of studying Parkinson's disease. In a new wing valued at two million dollars, such rats live alongside other rats that have been genetically modified using CRISPR. The cages are spotlessly clean, regulated in terms of temperature and populated with rats from floor to ceiling. To place an order it is only necessary to select the 20 suitable rats, carefully pack them in boxes and send them on a flight to California. The same process is also true for rats used in research on a variety of diseases from schizophrenia to pain control.
But if some customer needs a rat or mouse that is not in stock, the process is different. A Sage customer interested in investigating the link between Parkinson's and a new suspected gene, or even a particular gene mutation, has several options. The scientists at Sage can use CRISPR to disable the gene, create a mutation, or replace the gene with a human gene. Many diseases from Parkinson's and cystic fibrosis to AIDS are affected by different genetic variants. With the old method, it usually takes a year to generate in animals the entire complex array of sequential mutations needed to study such diseases. Unlike the editing methods that preceded it, CRISPR allows researchers to create many genetic changes in a cell at the same time and quickly and reduce the time needed to produce a transgenic animal to weeks.

The employees at Sage begin the process by preparing DNA adapted to a specific need, using a chemical kit and RNA that matches the DNA. In a petri dish, they mix the RNA with Cas9 and these connect to a chemical conjugate with the properties of a gene editor: the CRISPR tool. For about a week they test the operation of CRISPR in animal cells. For this purpose, they use a kind of desktop scanner that transmits electric currents that allow CRISPR to penetrate the cells. CRISPR starts working, cutting the DNA and creating small additions or omissions. Since CRISPR is not XNUMX% efficient, it makes cuts and creates mutations in some cells but not all. To test the effectiveness of CRISPR's performance, the scientists collect the DNA from the cells, combine them into one pool and reproduce the area around the said mutation site. After processing and testing the DNA, they look at the results on a computer monitor. The mutated DNA appears as a faint band, and the more DNA CRISPR cuts, the brighter the band becomes.

Then the process moves to the animal wing, where scientists use CRISPR to quickly produce genetically modified embryos and create mutated rodents. I followed biologist Andrew Brown as he worked the magic of Crisper. Armed with gloves and blue paper clothes: a gown, aprons and a puffy bonnet, he bent over a surgical microscope and drew a fertilized rat egg with a glass pipette. From there he transferred the egg to a larger microscope with robotic arms, released it into a drop of liquid on a glass carrier and sat down on a stool. With his right hand he operated a steering rod that moved a hollow glass needle against the wall of the cell.
Through the lens of the microscope, the two nuclei, one of each parent, look like small craters on the surface of the moon. Brown fiddled with the cell until one of the nuclei came close to the tip of the needle. He pressed the button of the computer mouse and the needle injected a tiny drop of liquid containing Crispr through the cell membrane. The core swelled like a fast moving flower blossom. With a bit of luck Brown created a mutated cell. The three Sage technicians repeat this process 300 times a day, four days a week.

When Brown finished injecting the fertilized rat egg, he aspirated it with a pipette and transferred it to a Petri dish housed in an incubator at body temperature. He eventually injected the transgenic embryo, and 30 to 40 others, into a rat that served as a surrogate mother. After 20 days, the rat will give birth to 5-20 pups and when the pups are ten days old, the scientists at Sage will take tissue samples to check which of them carry a transgenic gene.
"That's the exciting part," says Brown. "It is possible that only one out of 20 carries the change, and it is called the founding animal in our language. When you reach this stage, everyone celebrates." When you watch Sage scientists prepare RNA or inject embryos, you get the impression that the process is done with ease. In such processes, genetically engineered animals are obtained in many laboratories. Sage CEO David Smoller calls it editing gardens for "the masses."
A promise hidden in a duty perhaps a little dangerous
As CRISPR races forward toward commercial use, scientists and entrepreneurs are inventing new applications for the method, some of which border on the pretentious. It will be possible to correct the chromosomal abnormality associated with Down syndrome early in pregnancy, or restore herbicide sensitivity to resistant weeds, or bring extinct animals back to life. No wonder the idea scares some people. Alarmed commentators have warned that in our race to slay the malaria mosquitoes, cure Huntington's disease or engineer good babies, we could be creating harmful new "Jurassic Park"-style genes.

Think, for example, of the idea of ​​using CRISPR to bleach malaria mosquitoes. It's one thing to destroy the malaria parasite, but it's another thing entirely to destroy the carrier, says Todd Quicken, a bioweapons security analyst at the Woodrow Wilson Center for International Studies. According to him, if the goal is to eradicate malaria, a disease that infects 200 million people a year and kills 600,000, we must be careful and not create ten other problems. "We have to ask, 'Do we really want to do this?' And if the answer is 'yes', what appropriate system do we have, what kind of security measures?"

To the scientists' credit, they are making rapid progress in understanding the practical dangers expected of the CRISPR method and developing appropriate responses. In July 2014, when a team from Harvard published an article on the elimination of mosquitoes with the help of CRISPR, the scientists called for a public debate and began to propose technological and regulatory solutions to control GMOs that would get out of control. "CRISPR is spreading at such an extraordinary speed," says Jeannette Lenshoff, a bioethicist with the research group. "Many people have not heard of it but many are already using it. This is a new dynamic." As part of the Genomic Innovation Initiative at Berkeley, Dudna formed a group to discuss the moral implications of CRISPR's applications.
It is hard to see how ethical anxieties could dampen enthusiasm for Crisper. In June 2014, for example, researchers at MIT reported curing adult mice of tyrosinemia, a rare liver disorder caused by a mutation in one of the enzymes, by injecting CRISPR directly through the tail. Using three strands of guide RNA with Cas9 and the correct DNA sequence for the mutated gene, they were able to insert the correct gene into one of 250 cells in the mouse livers. One month after the treatment, the healthy liver cells flourished and eventually replaced a third of the damaged liver cells. That was enough to overcome the disease. In August, Kamel Khalili, a virologist at Temple University, and his team reported that they were able to eliminate the HIV virus that causes AIDS in several human cell lines.
For Khalili, who has been devoting all his efforts to AIDS research since the dark days of the 80s, Crisper is nothing short of revolutionary. Despite the enormous progress in the treatment of AIDS, all the drugs used today only control the virus and do not destroy it. But using CRISPR, Khalili's team completely removes the HIV copy from the cell's genome and turns infected cells into healthy ones. According to Halili, CRISPR can also protect a cell that is not infected. Vaccination through the combination of a sequence from the attacking virus is the exact same mechanism as Dudna and her team saw in primitive bacteria. You can call it a genetic vaccine. "If you had asked me two years ago, 'Can I accurately remove the HIV virus from a person's cell?' I would answer that this is an impossible task. Now we have done it," says Khalili, "this is the ultimate cure."

in brief

• Scientists knew how to change the genomes of living things as early as the 70s, but the tools at their disposal were imprecise and unsuitable for large scale. Because of this, many experiments were too difficult and expensive. Now, a new method called CRISPR can revolutionize genome editing. This method, based on the immune defense system of bacteria, is fast, cheap and much easier than the old methods.
  Researchers are already working on CRISPR-based treatments for diseases including HIV and schizophrenia. But because CRISPR makes it possible to make changes to the genomes of living things so easily, ethics researchers fear negative consequences.

on the notebook
Margaret Knox is a freelance writer and editor in Boulder, Colorado.

• Basics
  How does Crisper work?
  Bacteria use a weapon called a crisper to cut down invading viruses. Scientists can use the process to chop up DNA sequences they want to change. Unlike previous genome editing methods, the CRISPR method uses a single enzyme, Cas9, for all cutting purposes. All the researchers have to do is create a guide RNA molecule that will guide the enzyme to the target. It is infinitely easier to synthesize such RNA than to synthesize enzymes.
• Create a guide RNA that includes a part that matches the desired DNA sequence.
• Connect the guide RNA to the enzyme that cuts each target, Cas9, and thus create the CRISPR tool.
  CRISPR is injected into the target cell. The guide RNA finds the appropriate DNA in the genome.
• The Cas9 enzyme cuts the two strands of DNA so that the gene is neutralized or a section of genetically engineered DNA is inserted in its place and the gene is changed.
• More on the subject
  RNA-Programmed Genome Editing in Human cells. Martin Jinek et al. in eLife, Article no. 00471; January 29, 2013.
  Cas9 as a versatile tool for engineering biology. Prashant Mali et al. in Nature methods, Vol. 10, pages 957-963; October 2013.
  Cas9 Targeting and the CRISPR Revolution. Rodolphe Barrangou in Science, Vol. 344, pages 707-708; May 16, 2014.
  The RNA revolution. Christine Gorman and Dina Payne Maron, Scientific American Israel, August-September 2014; http://www.sciam.co.il/archives/8113
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The article was published with the permission of Scientific American Israel