# data[].attributes.body
Main idea:
Understandig gene editing
Arguments/facts:
- Understandig
- gene editing
- Cell
- Process

Dialogue:

Changing cell info
Summary:
Gene editing refers to the process of changing regions of cellular DNA. These changes are permanent and heritable, resulting in a newly engineered cell line.
Additionally, Horizon offers a wide selection of pre-made edited cell lines and expert genome engineering services.
APA
Gene editing

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Ready-to-go or tailored for individual needs

Find 1000's of validated knockout models, point mutations in cancer-relevant cell models, and "CRISPR-ready" stable Cas9 or dCas9-VPR expressing cell lines.

Or use our online tool to design your own cell line and get an instant quote and timeline for your next cell line engineering project.

# data[].attributes.attachment_link.description
Ready-to-go or tailored for individual needs Find 1000's of validated knockout models, point mutations in cancer-relevant cell models, and "CRISPR-ready" stable Cas9 or dCas9-VPR expressing cell lines. Or use our online tool to design your own cell line and get an instant quote and timeline for your next cell line engineering project.

# data[].attributes.body
Main idea:
Thermo editing
Arguments/facts:
- Reaserchers
- Thermo
- Scientific
- Control

Dialogue: Thermo editing with humans

Summary:
To help researchers in their quest to understand how the genome influences phenotype, we’ve developed a complete set of trusted solutions for every step in the gene editing workflow. Because every lab is unique, we offer a variety of genome editing solutions to meet your needs. Whether you want results fast, seek full control over every design step, or need help engineering cells to your specific requirements, we have solutions that fit.
APA
Unknown(2020,5 december) Thermo fisher https://www.thermofisher.com/es/es/home/life-science/genome-editing.html

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To help researchers in their quest to understand how the genome influences phenotype, we've developed a complete set of trusted solutions for every step in the gene editing workflow. Design your CRISPR-Cas9 system or TALEN constructs for precise cleavage, knock-in, and tagging, transfect them efficiently into cells, and validate the genotypic and phenotypic results. Our optimized, validated products, protocols, and tools work together to eliminate the trial-and-error phase and help you get answers faster and with less effort.

Because every lab is unique, we offer a variety of genome editing solutions to meet your needs. Whether you want results fast, seek full control over every design step, or need help engineering cells to your specific requirements, we have solutions that fit.
Download our genome editing resource guide

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Gene Editing | Thermo Fisher Scientific - US

# data[].attributes.attachment_link.description
To help researchers in their quest to understand how the genome influences phenotype, we've developed a complete set of trusted solutions for every step in the gene editing workflow. Design your CRISPR-Cas9 system or TALEN constructs for precise cleavage, knock-in, and tagging, transfect them efficiently into cells, and validate the genotypic and phenotypic results.

# data[].attributes.body
Main idea:
Genome editing is a way of making changes to specific parts of a genome.

Arguments/facts:
-Therapies
-Changes to specific parts of a genome
-Changing genomes de tret disease
-CRISPR

Dialogue:

How do people change genome
Summary:
Genome editing is a way of making changes to specific parts of a genome. Dozens of countries already prohibit any attempt to start a pregnancy with edited embryos, yet some scientists seem eager to proceed.
Any discussion of how we might use this technology in the future needs to consider the serious societal consequences of human genome editing.
APA

Center for genetics and society(n.d.)fetrived from:https://www.geneticsandsociety.org/internal-content/what-human-gene-editing

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Genome editing is a way of making changes to specific parts of a genome. Scientists have been able to alter DNA since the 1970s, but in recent years, they have developed faster, cheaper, and more precise methods to add, remove, or change genes in living organisms. Researchers are working to develop therapies that use gene editing to treat children or adults for a range of conditions, including sickle cell, hemophilia, and some forms of cancer and blindness.

Since 2015, a few laboratories have been experimenting with a far more controversial use of CRISPR: editing the genomes of early human embryos, eggs, and sperm. If edited embryos are used to start a pregnancy, the changes affect every cell in the body of any resulting child, that child's offspring, their offspring, and so on. Dozens of countries already prohibit any attempt to start a pregnancy with edited embryos, yet some scientists seem eager to proceed.

In November 2018, researcher He Jiankui from Shenzhen, China announced the birth of the first gene-edited babies: twin girls publicly referred to as Lulu and Nana. In a reckless and widely condemned experiment, He had edited the DNA of two embryos and used them to start a pregnancy. The babies were born prematurely and their current health status is unknown.

These utterly unethical experiments have pushed the issue of human genome editing to the forefront of media, scientific, and public discussion and debate. Any discussion of how we might use this technology in the future needs to consider the serious societal consequences of human genome editing. This includes examining the rise of vast economic inequalities and the resurgence of overt xenophobia and racism in many parts of the world. It also includes acknowledging our eugenic histories and the present-day systemic oppression of women, people of color, Indigenous people, LGBTQ people, and people with disabilities, particularly as they relate to reproduction and ideas about who is "fit" to reproduce.

Human genome editing is not just a scientific issue. It is a political and social justice issue that intersects with the concerns of multiple movements, including disability rights, LGBTQ rights, reproductive rights and justice, racial justice, environmental justice, and health justice. Read on to learn more about human genome editing and why everyone should have a say in the decisions we make about whether and how to use this powerful technology.

What is CRISPR?

CRISPR is a gene editing technology that allows scientists to make changes to the DNA of living organisms more precisely and inexpensively than before. CRISPR stands for clustered regularly interspaced palindromic repeats. These segments of DNA occur naturally in bacteria, where they store information that helps recognize invading viruses. Associated enzymes, such as Cas9, then cut viral DNA out of the bacterial genes.

Scientists discovered that they can adapt CRISPR-Cas molecules to search for a specific DNA sequence and cut precisely at that point not just in bacteria, but in plant, animal, and human cells, too. They can also provide a new DNA sequence for the cell to use when it repairs the cut.

CRISPR-Cas is often compared to the "find and replace" function in a word processor, but this metaphor of gene "editing" can make it sound more precise than it actually is. CRISPR sometimes mis-recognizes a DNA sequence that is similar to the one it's looking for and cuts in the wrong place, causing "off-target mutations." Other times it might cut in the right place, but cause mistakes, or "indels," where DNA is incorrectly inserted or deleted.

Gene Therapy: Changing genomes to treat disease

There are two distinct ways gene editing might be used in humans. Gene therapy, or somatic gene editing, changes the DNA in cells of an adult or child to treat disease, or even to try to enhance that person in some way. The changes made in these somatic (or body) cells would be permanent but would only affect the person treated. One way this is already being done is by editing a person's immune cells to help them better fight cancer. Clinical trials will soon be underway to use CRISPR to edit blood cells as a treatment for sickle cell anemia and other blood disorders. Gene therapy raises many of the same social and ethical issues as other high-tech medical treatments, including ethical research practices, safety and effectiveness, unequal access to expensive treatments, and how we allocate resources, but is widely supported as a promising way to treat disease.

Germline Editing: Changing the genomes of future generations

But there is a much more controversial way that human gene editing could be used. In germline modification, gene editing would change the DNA of embryos, eggs, or sperm. Because germline DNA is passed down to all future generations, any changes whether they had beneficial or harmful effects would be as well. Some have proposed that germline editing could be used to prevent inherited diseases, but this would carry unacceptably serious safety, ethical, and social risks. And it's unneeded, since we already have safe and effective ways to prevent passing on an inherited disease. People at risk can use preimplantation genetic diagnosis (PGD), a way to screen embryos created through in vitro fertilization (IVF) and select one that is unaffected; this allows parents to have a genetically related child without passing on an inherited disease. PGD certainly raises its own ethical questions, particularly around disability rights and justice, but it poses fewer safety and societal risks than germline editing would.

Understanding the Social and Ethical Risks

New technologies often raise ethical questions about their unknown risks and benefits. These questions become especially tricky - and essential - when we are talking about something like human germline editing, which affects future generations who obviously can't consent to the changes being made to their DNA. What risks would women (who are rarely mentioned in discussions about human gene editing for reproduction) be subject to as the ones who would carry pregnancies started with genetically modified embryos and deliver the resulting children (for themselves or for others)? How could potential parents make informed decisions when there would be unknown health risks that might emerge during pregnancy for the woman and the fetus, epigenetic effects, and health issues that might not develop until adulthood or old age (or even in future generations)? It would be extremely difficult, if not impossible, to ethically conduct the kind of follow-up studies that would be necessary to say that human genome editing is safe enough to use in reproduction.

But focusing on these obvious safety risks takes too narrow a view and overlooks the many serious social and ethical risks that germline editing would pose. Imagine wealthy parents being able to purchase enhancements (real or perceived) for their children, and the kind of world that would result if children's education and life chances were thought to be determined at birth by their DNA. Imagine the long-term consequences of imposing the preferences and biases we hold today on the genes of all future generations. Consider the potential effects on groups that have less power in society and are already discriminated against, including people with disabilities, people of color, and women. Ableism, racism, and reproductive injustices would likely be exacerbated by human genome modification, if it were ever allowed. These and other social inequalities that already shape our lives could rapidly grow worse, and new forms of inequality could be introduced, leading to a new form of eugenics.

While it might seem possible to avoid such dire outcomes by limiting the use of germline gene editing to the prevention of serious diseases, this would be extremely difficult. The line between therapy and enhancement is fuzzy and would be nearly impossible to enforce. How would we determine which diseases are serious enough to edit out? And who would decide? There are many disabled people who value their differences as a form of human diversity and do not think they need to be "treated" or " cured." Allowing just some uses of germline gene editing for reproduction would mean opening the door to all uses. For these reasons, over 40 countries have banned human germline modification.

Who Gets to Decide?

Human germline editing is not just a scientific or technical issue. It affects how we understand ourselves as humans and what kind of future we want to build. It has implications for society as a whole, not just individuals. Therefore, decisions about whether to permit germline modification should not be made by small groups of scientists or bioethicists, by biotechnology companies, or by wealthy elites. Human germline editing is an urgent social justice issue; we need public discussions of it that are open to all.

# data[].attributes.attachment_link.description
Genome editing is a way of making changes to specific parts of a genome. Scientists have been able to alter DNA since the 1970s, but in recent years, they have developed faster, cheaper, and more precise methods to add, remove, or change genes in living organisms. Researchers are working to develop therapies that use gene editing to treat children or adults for a range of conditions, including sickle cell, hemophilia, and some forms of cancer and blindness.

# data[].attributes.body
Main idea:
Tool for gene editing
Arguments/facts:
- Tool
- Cells
- Transformation
- Second effects

Dialogue:

These will be a better way of changing DNA
Summary:
Advances in gene editing, and CRISPR technology in particular, may soon make it possible. In the early days, CRISPR was used to simply make cuts in DNA. Today, it’s being tested as a way to change existing genetic code, even by inserting all-new chunks of DNA or possibly entire genes into someone’s genome. «Even if you start with a normal cholesterol level, and you turn off PCSK9 and bring cholesterol levels even lower, that reduces the risk of having a heart attack,» says Musunuru.
APA
Hamzelou, J. (2023, 20 enero). Next up for CRISPR: Gene editing for the masses? MIT Technology Review.

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Skip to Content

Last year, Verve Therapeutics started the first human trial of a CRISPR treatment that could benefit most people-a signal that gene editing may be ready to go mainstream.

CRISPR for high cholesterol is one of MIT Technology Review's 10 Breakthrough Technologies of 2023. Explore the rest of the list here.
We know the basics of healthy living by now. A balanced diet, regular exercise, and stress reduction can help us avoid heart disease-the world's biggest killer. But what if you could take a vaccine, too? And not a typical vaccine-one shot that would alter your DNA to provide lifelong protection?

That vision is not far off, researchers say. Advances in gene editing, and CRISPR technology in particular, may soon make it possible. In the early days, CRISPR was used to simply make cuts in DNA. Today, it's being tested as a way to change existing genetic code, even by inserting all-new chunks of DNA or possibly entire genes into someone's genome.

These new techniques mean CRISPR could potentially help treat many more conditions-not all of them genetic. In July 2022, for example, Verve Therapeutics launched a trial of a CRISPR-based therapy that alters genetic code to permanently lower cholesterol levels.

The first recipient-a volunteer in New Zealand-has an inherited risk for high cholesterol and already has heart disease. But Kiran Musunuru, cofounder and senior scientific advisor at Verve, thinks that the approach could help almost anyone.

The treatment works by permanently switching off a gene that codes for a protein called PCSK9, which seems to play a role in maintaining cholesterol levels in the blood.

"Even if you start with a normal cholesterol level, and you turn off PCSK9 and bring cholesterol levels even lower, that reduces the risk of having a heart attack," says Musunuru. "It's a general strategy that would work for anyone in the population."

CRISPR's evolution

While newer innovations are still being explored in lab dishes and research animals, CRISPR treatments have already entered human trials. It's a staggering accomplishment when you consider that the technology was first used to edit the genomes of cells about 10 years ago. "It's been a pretty quick journey to the clinic," says Alexis Komor at the University of California, San Diego, who developed some of these newer forms of CRISPR gene editing.

Gene-editing treatments work by directly altering the DNA in a genome. The first generation of CRISPR technology essentially makes cuts in the DNA. Cells repair these cuts, and this process usually stops a harmful genetic mutation from having an effect.

Newer forms of CRISPR work in slightly different ways. Take base editing, which some describe as "CRISPR 2.0." This technique targets the core building blocks of DNA, which are called bases.

There are four DNA bases: A, T, C, and G. Instead of cutting the DNA, CRISPR 2.0 machinery can convert one base letter into another. Base editing can swap a C for a T, or an A for a G. "It's no longer acting like scissors, but more like a pencil and eraser," says Musunuru.

In theory, base editing should be safer than the original form of CRISPR gene editing. Because the DNA is not being cut, there's less chance that you'll accidentally excise an important gene, or that the DNA will come back together in the wrong way.

Verve's cholesterol-lowering treatment uses base editing, as do several other experimental therapies. A company called Beam Therapeutics, for example, is using the approach to create potential treatments for sickle-cell disease and other disorders.

And then there's prime editing, or "CRISPR 3.0." This technique allows scientists to replace bits of DNA or insert new chunks of genetic code. It has only been around for a few years and is still being explored in lab animals. But its potential is huge.

That's because prime editing vastly expands the options. "CRISPR 1.0" and base editing are somewhat limited-you can only use them in situations where cutting DNA or changing a single letter would be useful. Prime editing could allow scientists to insert entirely new genes into a person's genome.

That would open up many more genetic disorders as potential targets. If you want to correct a specific mutation that is beyond the reach of base editing, "prime editing is your only option," says Musunuru.

If it works, it could be revolutionary. A hundred people with a disorder might have all kinds of genetic influences that made them vulnerable to it. But inserting a corrective gene could potentially cure all of them, says Musunuru. "If you can put in a fresh new working copy of the gene, it may not matter what mutation you have," he says. "You're putting in a working copy, and that's good enough."

Together, these new forms of CRISPR could dramatically broaden the scope of gene-editing treatments-making them potentially available to many more people, and for a much broader range of disorders. The target diseases don't even have to be caused by genetic mutations. In fact, even some of the older CRISPR approaches could be used to target diseases that aren't necessarily the result of a rogue gene. Verve's treatment to permanently lower cholesterol is a first example of a CRISPR treatment that could benefit the majority of adults, according to Musnuru.

Genetic vaccinations

Verve's approach involves swapping a base letter in the gene that codes for the PCSK9 protein. This disables the gene, so much less protein is made. Because the PCSK9 protein plays an important role in maintaining levels of LDL cholesterol-the type associated with clogged arteries-cholesterol levels drop too.

In experiments, when mice and monkeys were given the treatment, their blood cholesterol levels dropped by around 60 to 70% within a few days, says Musunuru. "And once it's down, it stays down," he adds. The company expects its first human clinical trial to run for a few years. If the trial is successful, the company will continue with larger trials. The treatment will have to be approved by the US Food and Drug Administration before it can be prescribed by doctors in the US. "It will be a while before any [CRISPR treatments] are actually approved for use," says Musunuru.

But in the future, he says, we might be able to use the same approach to protect people from high blood pressure and diabetes.

Komor of UC San Diego says a CRISPR-based treatment to prevent Alzheimer's might also be desirable. But she cautions that editing the genomes of healthy people is ethically ambiguous and could be an unnecessary gamble for people who are otherwise well. "If I was given the opportunity to do editing of my liver cells to reduce cholesterol potentially in the future, I would probably say no," she says. "I want to keep my genome as is, unless there's a problem."

Any new treatment has to be at least as safe as what is already available, says Tania Bubela, who studies the legal and ethical implications of new technologies at Simon Fraser University in Burnaby, British Columbia. Plenty of drugs have side effects. "The difference is that with a drug, you can ... change the person's medication," says Bubela. "With a gene therapy, I can't see how you would do that."

The price, as well as the safety, of any gene-editing treatment will determine whether it can really help the masses, Bubela says: "I find it difficult to believe that a gene-based therapy like CRISPR will ever be either safer or more cost-effective than a very simple cholesterol pill." But she accepts that these treatments could become cheaper, and that the "one-shot" approach might appeal to some.

There's a good reason the first trials of CRISPR have focused on people with rare disorders who have few options, says Komor: "Those are the people most in need." While broadening the applications of CRISPR is exciting, she says, "we have an ethical obligation to help those people before we help the general masses."

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# data[].attributes.attachment_link.title
Next up for CRISPR: Gene editing for the masses?

# data[].attributes.attachment_link.description
Skip to Content Last year, Verve Therapeutics started the first human trial of a CRISPR treatment that could benefit most people-a signal that gene editing may be ready to go mainstream. CRISPR for high cholesterol is one of MIT Technology Review's 10 Breakthrough Technologies of 2023. Explore the rest of the list here.

# data[].attributes.body
Main idea:
This web page talks about kids allowing other people to hack dna.
Arguments/facts:
-Hack
-Dna
-Online
-Talk

Dialogue:

Why do parents allow their kids to talk with strangers
Summary:
This web page talks about a conversation about kids talking online with other people.The strange people talks with the kids because they just want to hack
APA

Sneed, A. (2017, November 2). Mail-Order CRISPR Kits Allow Absolutely Anyone to Hack DNA. Scientific American. retrived from:https://www.scientificamerican.com/article/mail-order-crispr-kits-allow-absolutely-anyone-to-hack-dna/

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"We aren't going to get sick, are we?" my roommate Brett asked me. He cringed as I knelt down and stuffed a plate of E. coli bacteria-which came as part of the DIY CRISPR-Cas9 kit I bought online-into our fridge next to cartons of eggs, strawberry jam, bottles of beer and a block of cheese.

"No, we won't. The label says 'non-pathogenic,'" I replied, trying to sound assuring. But honestly, I had no clue what I was doing. I nudged all the food up against the fridge wall, and left a two-inch border around the plate of living cells-a no man's land between the microbes and our dinner. A couple inches probably would not stop the bugs, but I figured it couldn't hurt.

CRISPR-Cas9 (or CRISPR, for short) has given scientists a powerful way to make precise changes to DNA-in microbes, plants, mice, dogs and even in human cells. The technique may help researchers engineer drought-resistance crops, develop better drugs, cure genetic disorders, eradicate infectious diseases and much more. Ask any biologist, and they'll likely tell you that CRISPR is revolutionary. It's cheap and effective, and in many cases, it works much better than older methods for making genetic modifications. Biologists will also tell you that CRISPR is very easy to use. But what does "easy to use" mean?

I am not a DIY scientist, much less a professional scientist. You won't find me swabbing my cheek cells for DNA or tinkering with yeast in a lab on the weekend. But I wondered: Is CRISPR so easy thateven amateurs like me can make meaningful contributions to science? And also, does this new technique make gene editing so accessible that we need to worry about DIY scientists cooking up pandemic viruses in their basements? If you Google 'DIY CRISPR,' stories such as "What Happens If Someone Uses this DIY Gene Hacking Kit to Make Mutant Bacteria?" pop up.

I attempted to find answers to all these questions myself, starting with the plate of bacteria in the kitchen of my San Francisco apartment.

CUT AND SPLICE

CRISPR stands for "clustered regularly interspaced short palindromic repeats." The CRISPR system is made up of two components: a protein called Cas9 and a guide RNA,a string of nucleic acid molecules with a certain genetic code. Put them together, and they create a tool you can use to tweak an organism's genome. To do this, CRISPR searches the organism's DNA for a certain sequence-specifically, the one encoded by the guide RNA, which holds the inverse sequence of your target DNA. "Cas9 opens up the DNA, it separates the strands of the double helix in a very small area, and allows the guide RNA to pair with one of the strands," explains Dana Carroll, a professor of biochemistry at the University of Utah. "If it is a good match, cutting occurs. If it is not a good match, Cas9 and [the] guide RNA fall off and try again somewhere else." When it finds the right sequence, the Cas9 protein slices the DNA at that precise spot.

At this point, if you leave the cell alone, it will usually mend CRISPR's cut-but it will occasionally also make a mistake in the repair process, breaking a gene or other parts of the genome. Since CRISPR repeatedly goes back and slices the DNA again after the cell mends it, the gene eventually breaks, or, in technical terms, gets knocked out. And, if you add new DNA, the cell may incorporate it while fixing the cut. This means you can insert DNA where you want to in the genome-you just need to know the organism's genetic sequence of your desired target area.

Scientists originally discovered this sophisticated system in archaea and bacteria, which deploy CRISPR to chop up invading viruses. But a few years ago, researchers figured out how to repurpose CRISPR to run inpretty much any living thingthey want. And now it's made genetic engineering easier than ever before.

For my own experiment, everything I needed came in a small cardboard box-an assortment of bottles, tubes, plates, powders and liquids (plus E. coli). I ordered my kit for $130 from the crowd-funding site Indiegogo as part of a campaign created by Bay Area biohacker Josiah Zayner. Zayner has a PhD in molecular biophysics and spent two years as a research fellow at NASA. He ran the crowdfunding campaign out of his apartment, and by the end of it, he had raised over $70,000 and sold 250 DIY CRISPR kits-one of which now sat on my kitchen table. Zayner has now soldover a thousand kits, largely on his company's website, The Odin.

The idea behind the kit experiment is quite simple. The goal: modify the E. coli so that it can grow on an antibiotic called streptomycin, which normally kills bacteria. With materials and instructions from the kit, I will introduce CRISPR into the bacteria cells, and use it to rewrite a tiny part of their DNA, creating genetically altered cells that happily thrive on streptomycin. In the end, CRISPR will track down and then change only a single base pair (which are the building blocks for DNA)out of the 4.6 million base pairs in the E. coli genome. It will swap out the chemical compound adenine for cytosine-or, in terms of the genetic alphabet, an "A" for a "C." Because of that tiny code change, my bacteria cells will make the amino acid lysine instead of another one, threonine. If my gene editing succeeds, this will stop streptomycin from interfering with the E. coli.

On a Monday afternoon in May, I donned latex gloves and spread newspaper over the dinner table. I grabbed three small plastic tubes from the freezer, picked up a pipette-a hand-held instrument, used in labs to measure out liquids-from my DIY kit, and started adding the CRISPR ingredients to tubes of E. coli.

I put in the Cas9 protein and guide RNA,which came as liquids in small, plastic test tubes,to my bacteria cells. Then I dipped my pipette into a small tube of DNA and tried to suck up 10 microliters. Nothing came out. I squinted at the tiny drop of clear liquid, and realized it had frozen solid. I had no clue why. Uh oh, I hope that's not a problem. Would it harm my experiment? I had absolutely no idea, so I just waited for the DNA solution to thaw and then squirted it into the bacteria tube. After several more steps, I spread my CRISPR'd bacteria onto three plastic plates and put them in my laundry room. The instructions said to wait 24-48 hours, then check for small white dots of bacteria. If I saw dots, CRISPR had done its job of splicing in the streptomycin-resistance gene. If not ... well, failure is also part of the scientific process.

I had no problem conducting the experiment- CRISPR is easy, I concluded. I basically just measured, scraped and stirred a bunch of ingredients, occasionally cooling them or heating them up. But for all the godlike powers that I imagined CRISPR gave me, I actually had little say over what I did to my bacteria. Everything was predetermined, with instructions laid out for me like steps in a cookbook: "Add 100 microliters Transformation mix to a new centrifuge tube," "Incubate this tube in the fridge for 30 minutes," and so on. Ultimately, I had made zero decisions. Of course, I could have designed a custom-made CRISPR experiment-but it would have taken more time, more materials, more money, and a lot more knowledge than I currently had.

I inspected my bacteria 48 hours later. Crossing my fingers, I lifted the lid on the first plate. No white dots. Then the second plate: nothing. My stomach sank with disappointment. Then I gently raised the lid off the third plate, and saw ... something. The plate had two faint milky white circles. Had CRISPR worked? Maybe. But then why did one plate have white spots, but not the other plates? I'd followed the same steps for each of them. Maybe my mind was playing tricks on me. Or perhaps I had contaminated the third plate. If only I could show my plates to someone who knew how to interpret them. Like a scientist. Unfortunately, there was no one to ask in my kitchen.

OPEN TO EVERYONE

A few weeks later, I drove 40 miles south of San Francisco to meet a DIY scientist named Johan Sosa, who knows way more about CRISPR than I do. We met at BioCurious, a community laboratory in Sunnyvale where he works most weekends and some evenings. Located in Santa Clara, BioCurious is a co-working space outfitted with science equipment and shared by "scientists, technologists, entrepreneurs, and amateurs who believe that innovations in biology should be accessible, affordable, and open to everyone," according to its Web site.The lab is funded by donations and members-Sosa is one of itsseveral dozenmembers.

At six foot, five inches, Sosa towers over most people. "I'm probably the tallest DIY biologist," he jokes. He laughs easily, which offsets his height to lend him a gentle, laid-back manner. He is 40 years old, his dark hair flecked with silver. Originally from Sri Lanka, Sosa came to the U.S. for college at 15 years old to study computer science, and he's worked for Bank of America and IBM as a computer security specialist and software engineer. He now has a day job in computer security-but he spends most of his free time at BioCurious. "You could say I have no life," he chuckles, "This is my biggest hobby." He has learned everything he knows about science (both theory and lab techniques) from others at BioCurious, by reading science papers, watching YouTube videos, attending lectures, and also through trial-and-error in his own research.

Sosa is one of a few DIY scientists at BioCurious using CRISPR. He first read about the technique in 2012, from one of biochemist Jennifer Doudna'spapers in the journal Science. Doudna, a professor at University of California, Berkeley, is one of the pioneers of CRISPR. "I didn't think it was that big of a deal, because I knew there were already other ways of modifying DNA," he recalls, "But I did think, 'This is something I could do.'" He started experimenting with CRISPR in 2013.

On a muggy, overcast afternoon, I followed Sosa inside BioCurious. We strolled through a lobby and into a large windowless room. It had big cabinets crammed with bottles of liquid, racks of latex gloves, a giant bio-hood, microwave and fridge. Microscopes, scales, centrifuges and a bunch of other well-worn science equipment lay scattered across lab desks. A calm thrum of buzzing machinery filled the room, and test tubes shook quietly in an incubator nearby. Johan walked around the room, searching for a thermometer. "One thing about a DIY lab is that you leave something somewhere, and it always ends up somewhere else," he told me.

I had joined Sosa at BioCurious so I could learn more about what CRISPR means for DIY scientists, and also to do a (hopefully) more successful experiment, with his help. We decided on a very basic goal: we would use the powerful editing tool to cut DNA he had already extracted from yeast cells. This task is easier than the one I tried in my kitchen, because you don't need to get CRISPR inside cells in order to slice the DNA. Professional scientists might use such a method as an intermediate step, such as when they need to cut and paste DNA together to make a gene as part of a bigger research project. "That's a type of experiment everyone does every day," explains Charles Gersbach, a professor of biomedical engineering at Duke University-though he notes that traditionally, researchers have used a type of protein called a restriction enzyme, not CRISPR, to do this.

Sosa and I pulled on latex gloves and carefully pipetted liquids into tubes to make our guide RNA from scratch-we first synthesized DNA strands with a specific sequence we wanted, used that as a template for the RNA, then destroyed the DNA and isolated the guide RNA from our test tube mixture. Later, we put the RNA in a new test tube, along with the other materials needed to make CRISPR work in this experiment: protein buffer, bovine serum albumin (a protein isolated from cows),water. Sosa sucked up the yeast DNA in his pipette. Without warning, the plastic needle-nose tip fell off into the tube of DNA. Someone, he told me, had donated the tips to their lab, but they weren't the correct size. "I guess you got the full DIY experience," he smiled, and pushed the plastic tip back onto the pipette. Then he picked up the Cas9 protein. "Here's the world-famous Cas9," he said, and handed it to me. I added it to our test tube.

Until CRISPR, DIY scientists didn't have an easy, cheap or reliable way to precisely edit DNA. Many of them couldn't afford the pricey and imperfect tools that professional scientists used for gene editing at the time. "Before CRISPR, there was TALENS [transcription activator-like effector nucleases] and zinc finger nucleases-older technologies that were not as precise or reliable," explains Sosa. "They were out of the budget and the time constraints of DIY scientists." Sosa says that if a DIY-er used those other technologies, it might cost him or her thousands of dollars to do a genetic engineering experiment. But with CRISPR, it's vastly more affordable, especially if you want to attempt an experiment more than once. "With TALENS, you try it once and fail," says Sosa. "With CRISPR, you can try it multiple times. That alone is a big deal."

This means that CRISPR gives DIY-ers a whole new way to do science. So far, Sosa and his lab mates have tried out CRISPR in a number of ways: cutting yeast genomes, slicing DNA inside E. coli cells, and attempting to modify the CRISPR system by shrinking it or attaching other molecules to it. Sosa has goals for his CRISPR research. "I want to understand how a cell really functions, and what are all the little things that happen in it," he explains. "And when something goes wrong [such as in diseases], how to fix it or make it do what I want."

After several hours, Sosa and I checked to see if CRISPR had cut our yeast DNA. We dyed our DNA-CRISPR mixture blue and ran it through an electrically charged gel, which separates bigger DNA pieces from smaller ones. Tiny channels in the gel run from one charged end to the other, and the sliced DNA strands are pulled through them towards the positively charged side. If our experiment succeeded, we should see two blue bands for the short CRISPR-cut DNA strands in one spot, and one blue band for a longer, uncut piece of DNA (our control) in another location.

Sosa carried the gel into the bathroom, where we turned off the lights and looked at it under blue light. I held my breath while I inspected the gel for markings. One light-blue band gleamed in the dark-the control-and another single band lit up the spot where we should have seen our CRISPR'd DNA. "I don't know what happened, but it doesn't look right," Sosa said, "I don't think it worked."

I left the lab feeling defeated, and headed back to San Francisco. Sosa texted me a few minutes later.

Hey, I figured out what happened. There was no DNA to start with, he wrote.

What happened? I texted back.

I think the DNA had either degraded or gotten too diluted, he wrote.

Even if we had got all the other parts (RNA, proteins, etc.) working, it didn't matter. We hadn't given CRISPR any DNA to cut. My second attempt at CRISPR had utterly failed.

BETTER, FASTER, CHEAPER

My own frustrating struggles with CRISPR aside, I wanted to see what professional biologists are doing with CRISPR, so I visited the lab of Nipam Patel at the University of California, Berkeley. After a quick tour of the lab, I sat down and stared into the microscope at a small, writhing marine creature: Parhyale hawaiensis, commonly called a beach hopper. At one centimeter long, Parhyale looks puny-you'd step on it at the beach without even noticing. But under the microscope, this female hopper resembled a giant translucent shrimp with many powerful, kicking legs. Parhyale is the star of this lab. "We're looking at how you develop an individual body," explains Erin Jarvis, a PhD student in Patel's lab, "And also how you build a body form over evolutionary time." And they're using CRISPR to do it.

With CRISPR, these researchers knock out so-called Hox genes in Parhyale. Hox genes are found in all animals, including humans, and they control the development of their body plans. Among other things, they determine what appendages-such as swimming legs, claws and antennae-grown on which section of the body.Knock out a certain Hox gene with CRISPR and Parhyale will grow forward-walking legs where it should have jumping legs, for example.

Parhyale has nine Hox genes, and Patel's team has knocked out seven of them. The researchers also have plans to add completely new genes to Parhyale using CRISPR-they've already done it once, by inserting a gene that codes for green fluorescent proteins, which allowed the researchers to visualize where a specific Hox gene is expressed in Parhyale."From an evolutionary perspective, this [gives] us insight into how body plans evolve between species," when comparing Parhyale to, for example, the well-studied fruit fly, Drosophila, explains Patel. "We believe that such evolutionary patterns help us understand the general mechanisms by which evolution creates animal diversity.... What we learn improves our knowledge about the function of these genes in other animals, including humans."

CRISPR has transformed how Patel and his colleagues do their research. His lab has looked at Parhyale for about 20 yearsnow. Before CRISPR they used another technique to knock out genes that required a lot more money, and even then, it wasn't very efficient. It cost them about $900 to knock out a single genein a group of Parhyale embryoswith their other method. The technique, called "RNA interference," silences expression of a gene-it doesn't genetically knock it out as CRISPR does. The problem was, sometimes the method didn't work at all.

Now it costs them less than $100 to knock out a gene. "Suddenly, with CRISPR, you don't have to decide, 'Which one gene do I want to put all my resources into?'" Jarvis says, "You can try a lot of different genes." And with CRISPR, they're able to break genes in up to 75 percent of Parhyale embryos, versus a maximum 25 percent success rate with the old technique. Even better, they now have the ability to mutate several genes at once with CRISPR, which means they can now see how genes interact. When the researchers had tried to mutate multiple genes with their old technique, it rarely worked. (Though Patel notes that the older RNA technique is still very useful for certain applications).

Their research takes less time with CRISPR, too-in a study Patel's lab published in Current Biology in 2015, they knocked out six Hox genes in about a year. Before that, they had already spent yearstrying to break a specific Hox gene with their old method, but were never able to do it. "Everything just goes faster," says Patel, a professor of genetics, genomics and development. "CRISPR-Cas9 is an incredibly elegant system, and it's very easy to control." It also makes it simpler to study more exotic creatures (beyond the standard flies and mice), such as animals like Parhyale or butterflies. "It's always been hard to work with a new organism," says Jarvis, "CRISPR is awesome because suddenly, you don't have to spend decades developing a model." As long as you have the sequence of the gene you want to target, you're set.

Patel's lab is hardly the only one capitalizing on CRISPR-scientists around the world are exploring all sorts of different uses for the gene editing tool, like wiping out malaria-spreading mosquitoes, finding new ways to treat cancer, or engineering disease-resistant crops. In July, researchers announced they had successfully edited the genome of viable human embryos with CRISPR; the technique allowed them to fix a disease-causing mutation in the embryos' DNA (though some are now skeptical of the researchers' results). Just a few weeks later, scientists in Massachusetts reported they had made a significant advance towards pig-to-human organ transplants. They used CRISPR to inactivate 25 viruses intrinsic to pigs' genomes, overcoming a big obstacle in making porcine transplants safe for humans.

PLAYING GOD

I had reached the end of my CRISPR experiment-so what had I learned? First, I found out it was not completely crazy for my roommate to wonder whether a DIY CRISPR kit in our fridge would make us sick. This year, German authorities restricted imports of the Odin DIY CRISPR bacteria kit after the Bavarian Health and Food Safety Authority tested two kits and found them to contain potentially pathogenic bacteria. But even theEuropean Center for Disease Prevention and Control concluded that there was little to worry about-that "the risk of infection by the contaminating strains in the kit is low for the users ... assuming that they are healthy people." (Zayner declined to comment on the record about the incident, but he publicly posted a response on Twitter, where he criticized the methodology used by the Bavarian agency and denied wrongdoing by his company. The kits are still available for purchase online through The Odin.)

As for my bigger question-could untrained DIY-ers actually achieve scientific breakthroughs?-I asked academic researchers what they thought. Dana Carroll, for his part, believes amateurs could make meaningful discoveries. "In the professional science community, people keep coming up with new ways to use this technology-people are really only limited by their imagination," he explains. "It's possible that people working in their garages or their kitchens will come up with a novel application or a solution to a problem that professionals just haven't gotten around to." And Carroll says it would be easy for a DIY-er to share any discoveries with researchers, by attending their talks or simply by contacting them through their Web sites. Yet he notes that the DIY community faces limitations, because amateur scientists likely would lack the necessary resources. "It's unlikely they will bring a major application all the way to fruition," he says, "But they could certainly get started on something."

Finally, what about the nightmare scenario: Is CRISPR so easy to use that we need to worry about biohackers-either accidentally or intentionally-creating dangerous pathogens? Carroll and others think that the danger of putting CRISPR in the hands of the average person is relatively low. "People have imagined scenarios where scientists could use CRISPR to generate a virulent pathogen, " he says. "How big is the risk? It's not zero, but it's fairly small." Gersbach agrees. "Right now, it's difficult to imagine how it'd be dangerous in a real way," he explains, "If you want to do harm, there are much easier and simpler ways than using this highly sophisticated genetic editing technique."

Back in Patel's lab, Jarvis replaced the squirming female beach hopper under my microscope with a tiny Parhyale embryo. Jarvis told me she knocked out a Hox gene called Abd-B in this one-the embryo will grow jumping legs where it should have swimming legs, and forward walking legs instead of anchor legs. At this point, it just looked like an opaque ball of goo to me.

Next to me, another grad student examined a fragment of a brown and goldbutterfly wing-Patel's lab is also knocking out butterfly genes with CRISPR to see how they build wing color. "An old grad student used to joke that we were genetically modifying the wings to make the Mona Lisa," Jarvis told me. I laughed and glanced back under the microscope. A puff of my breath suddenly struck the Parhyale embryo. It danced wildly around the petri dish, like a grain of sand caught in a windstorm.

# data[].attributes.attachment_link.title
Mail-Order CRISPR Kits Allow Absolutely Anyone to Hack DNA

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"We aren't going to get sick, are we?" my roommate Brett asked me. He cringed as I knelt down and stuffed a plate of E. coli bacteria-which came as part of the DIY CRISPR-Cas9 kit I bought online-into our fridge next to cartons of eggs, strawberry jam, bottles of beer and a block of cheese.

# data[].attributes.body
Main idea:
Gene editing teching.
Arguments/facts:
- 2016
- DNA
- Lab
- 13 years old kid

Dialogue: Experimenting with kids is not a good thing bud they offer them self to doit thats why is not a bud thing.

Summary:
The paper, published by David Liu’s lab at the Broad Institute of MIT and Harvard, described a version of Crispr gene editing that allowed for more precise changes than ever before. «It seemed like science fiction had arrived,» says Qasim, who teaches at University College London.
APA
Mullin, E. (2022, 23 diciembre). A More Elegant Form of Gene Editing Progresses to Human Testing. Retrived from https://www.wired.com/story/a-more-elegant-form-of-gene-editing-progresses-to-human-testing/

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In April 2016, Waseem Qasim, a professor of cell and gene therapy, was captivated by a new scientific paper that described a revolutionary way to manipulate DNA: base editing. The paper, published by David Liu's lab at the Broad Institute of MIT and Harvard, described a version of Crispr gene editing that allowed for more precise changes than ever before. "It seemed like science fiction had arrived," says Qasim, who teaches at University College London.

The genetic code of every living thing is made up of a string composed of four chemical bases: A, C, G, and T. These pair up to form the double helix structure of DNA. Traditional Crispr and previous gene editing methods work by cutting DNA's double-stranded helix in order to knock out a disease-causing gene, for instance. Base editing, on the other hand, simply swaps one chemical base for another in order to correct a mutation or disable a gene. The first base editor that Liu's lab described could convert a C to a T. Others have been invented since.

Scientists immediately recognized the value of base editing. Many inherited diseases, such as cystic fibrosis and sickle cell anemia, are caused by single-base changes in DNA. Now those mutations could, in theory, be fixed by converting one base for another. Qasim and his team wanted to use base editing for another purpose: altering immune cells in an attempt to treat cancer.

Using Liu's paper as a guide, Qasim and his team created their own base editors and found that they were incredibly efficient at making genetic changes to cells in the lab. Over the next six years, they worked to improve the technology, and in May, they put it to the ultimate test, using it to treat a leukemia patient in hopes of curing her cancer. It was the first time this new form of gene editing was used to treat a human being.

The patient, a 13-year-old named Alyssa, was diagnosed with a rare and aggressive type of cancer called T-cell leukemia in May 2021. An important part of the immune system, T cells normally protect the body from infection. But in T-cell leukemia, they grow uncontrollably. Doctors tried to treat Alyssa with chemotherapy and a bone marrow transplant, but her cancer came back.

With no other treatment options left, Alyssa was eligible for a trial testing the experimental base editing therapy. Qasim and his team collected T cells from a healthy donor and used base editing to make four separate changes-all C to T base conversions-to the cells. The edits allowed the donor T cells to slip past the body's defenses, recognize a certain receptor on leukemia cells, and kill the cancer. Doctors at Great Ormond Street Institute of Child Health, part of University College London, then infused the edited cells into Alyssa's bloodstream.

After receiving the edited cells, Alyssa experienced an inflammatory side effect known as cytokine release syndrome, a common side effect with cancer immunotherapy. In some patients, it can be life-threatening, but Alyssa's symptoms were mild and she recovered quickly, Qasim says. A month after her infusion, her cancer was in remission, and she continues to do well. "We've confirmed the disease levels are still undetectable," Qasim says. He presented these preliminary results earlier this month at the American Society of Hematology meeting in New Orleans. (The findings have not yet been published in a peer-reviewed journal.)

# data[].attributes.attachment_link.title
A More Elegant Form of Gene Editing Progresses to Human Testing

# data[].attributes.attachment_link.description
In April 2016, Waseem Qasim, a professor of cell and gene therapy, was captivated by a new scientific paper that described a revolutionary way to manipulate DNA: base editing. The paper, published by David Liu's lab at the Broad Institute of MIT and Harvard, described a version of Crispr gene editing that allowed for more precise changes than ever before.

# data[].attributes.body
Main idea:
Human genome editing
Arguments/facts:
- Method
- Changes
- Genome
- Interventions

Dialogue:
Interventions of bad use of gene editing.

Summary: Genome editing is a method for making specific changes to the DNA of a cell or organism. It can be used to add, remove or alter DNA in the genome. Application of somatic human genome editing has already been undertaken, including in vivo editing, to address HIV and sickle-cell disease, for example. Although somatic human genome editing is well established and acceptable for treatments in some scientifically advanced countries with regulations in place, challenges remain. Other challenges are associated with rogue clinics, medical travel, as well as the reporting of illegal, unregistered, unethical or unsafe research and other activities including the offer of unproven so-called therapeutic interventions.
Heritable human genome editing refers to editing of nuclear DNA in a way that may be heritable across generations.

APA
Human genome editing. (2021, 20 octubre).

# data[].attributes.body
Main idea:
Gene Editing – Digital Media Kit
Arguments/facts:
- Genome Editing in Research
- Genome Editing in the Clinic
- Ethical and Safety Concerns

- Genome Editing and NIH Funding

Dialogue:

They are making clinics of gene editing.
Summary:
Genome editing can be used to correct, introduce or delete almost any DNA sequence in many different types of cells and organisms. While techniques to modify DNA have existed for several decades, new methods have made genome editing faster, cheaper and more efficient. Genome editing builds on an earlier discovery that a broken section of DNA in a gene triggers a cell’s repair mechanism to stitch together the break. Genome editing allows researchers to mimic this natural process of DNA repair.

Advanced genome editing methods engineered from proteins include zinc-finger nucleases , transcription activator-like effector nucleases , and meganucleases. CRISPR/Cas9 is the most widely used genome editor and is a powerful tool for understanding gene function.
APA
Gene Editing â Digital Media Kit. (2020, 5 noviembre). National Institutes of Health (NIH).

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Genome editing, also called gene editing, is an area of research seeking to modify genes of living organisms to improve our understanding of gene function and develop ways to use it to treat genetic or acquired diseases. Genome editing can be used to correct, introduce or delete almost any DNA sequence in many different types of cells and organisms. While techniques to modify DNA have existed for several decades, new methods have made genome editing faster, cheaper and more efficient. Genome editing builds on an earlier discovery that a broken section of DNA in a gene triggers a cell's repair mechanism to stitch together the break. Genome editing allows researchers to mimic this natural process of DNA repair.

Advanced genome editing methods engineered from proteins include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases. An additional method is called clustered regularly interspaced short palindromic repeats, also known as CRISPR/Cas9.

CRISPR/Cas9 is the most widely used genome editor and is a powerful tool for understanding gene function. Because CRISPR/Cas9 is an RNA-based system, it can be more efficiently and easily modified than the protein-based approaches and allows for targeting of multiple sites. CRISPR was discovered through NIH-funded basic research on how bacteria defend themselves from viruses. CRISPR/Cas9 works by cutting a DNA sequence at a specific genetic location and deleting or inserting DNA sequences, which can change a single base pair of DNA, large pieces of chromosomes, or regulation of gene expression levels.

Genome Editing in Research

Genome editing is widely used in studies in a variety of organisms. For example, CRISPR is used to make "knockout" models of disease in a wide range of animals, enabling researchers to study the underlying genetic causes. It also is being used to change genes in certain tissues or organs, facilitate the study of diseases by focusing on culprit genes, create cell models of disease such as in human pluripotent stem cells and inactivate viruses in pigs so that pig organs could potentially be used as a source of replacement organs for humans. It also is being explored to modify yeast cells to make biofuels and to improve strains of agricultural crops. Gene drive technology using CRISPR allows for the spread of engineered traits through populations of sexually reproducing organisms at a rate more rapid than what occurs in natural evolution. This research could be applied to alter mosquito populations to help interfere with the transmission of infectious diseases.

Genome Editing in the Clinic

NIH supports human gene therapy research, including genome editing approaches in somatic cells, for a wide array of diseases and conditions with grants, contracts and targeted efforts, such as the Somatic Cell Genome Editing Program. Somatic cells are any cells not involved in human reproduction. This means that changes in somatic cells are not inherited by subsequent generations. CRISPR and other gene editing methods, especially ZFNs, are speeding gene therapy approaches to treat many human conditions. In 2014, the first clinical application of genome editing involved the use of ZFNs to make human cells resistant to HIV-1 by disrupting a gene required for the virus to infect cells. In 2017, a clinical trial testing ZFNs to correct Hunter syndrome (MPS II) was launched. Caused by an enzyme deficiency, Hunter syndrome can cause abnormalities in the skeleton, heart and respiratory system. The clinical trial was the first genome editing approach administered directly to research participants. TALENs are being studied in T cell immunotherapy approaches to create "off-the-shelf" universal donor T cells that don't have to be developed for each cancer patient. Genome editing approaches are also being pursued as part of NIH's Cure Sickle Cell Initiative, and CRISPR is being used as a diagnostic tool to detect viruses such as Zika and dengue. In October 2019, NIH and the Bill and Melinda Gates Foundation announced a collaboration to support studies to advance the development of gene-based approaches to cure sickle cell disease and HIV.

Ethical and Safety Concerns

Applications of genome editing have raised scientific, safety, ethical and policy concerns. NIH does not fund any use of genome editing in human embryos. The Dickey-Wicker amendment prohibits the use of Congressionally appropriated funds for the creation of human embryos for research purposes or for research in which human embryos are destroyed. Additionally, serious and unquantifiable safety and ethical issues presented by altering the germline in ways that affect the next generation without consent, and a current lack of compelling medical applications, do not justify the use of genome editing in embryos. In 2017, the National Academies of Science, Engineering, and Medicine (NASEM) issued a report, Human Genome Editing: Science, Ethics, and Governance (2017), which recommended that clinical trials using gene editing in embryos should be permitted only within a robust and effective regulatory framework and only when certain criteria are met. However, in November 2018, Chinese scientist He Jiankui announced that he had used the CRISPR/Cas9 technique to create the first genome edited babies, claiming to have disabled copies of a gene in the embryos to confer HIV resistance. This work raised serious questions of medical necessity, off-target effects and the potential for susceptibility to other infections. It led to nearly universal condemnation by the international scientific community, and the NIH Director issued a statement expressing serious concerns about the claim and urging continued international dialogue. On March 14, 2019, the NIH Director issued a statement supporting the call for an international moratorium on research establishing human pregnancies using germline-edited embryos. NIH continues to be involved in ongoing efforts by the World Health Organization and sponsored a study of the NASEM/Royal Society of the United Kingdom to examine the issues associated with human gene editing and develop global governance and oversight approaches. As discussed in the Director's blog, the international commission concluded in the report, Heritable Human Genome Editing (link is external) released in September 2020, that clinical use of heritable human gene editing should not be considered until it has been established that it is possible to make precise genomic changes efficiently and reliably without undesired changes in human embryos. If permitted, use should be limited to serious monogenic diseases. The report also included recommendations for scientific governance and oversight including the establishment of an international scientific advisory panel.

In many cases, the safety and bioethical concerns raised by the use of CRISPR/Cas9 in somatic gene therapy are like those first discussed at the emergence of recombinant DNA technology and human gene therapy. These concerns are well addressed in current oversight structures. For instance, in the United States, well-established oversight frameworks and the U.S. Food and Drug Administration's (FDA) regulatory authority over human gene therapy trials would apply to clinical research involving genome editing of somatic cells in humans. Additionally, biosafety guidance for the conduct of genome editing research is provided by the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules.

The potential effects of gene drives on individual species and the environment have also raised biosafety, biosecurity and ethical concerns. A NASEM report, Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values (2016), which was partially funded by NIH, explored the responsible conduct of gene drive research. In a 2016 statement by the NIH Director, NIH supported the report's recommendations to continue basic and applied research on gene drives, hold off on current release of gene drive-modified organisms into the environment, have funders coordinate to encourage responsible development of gene drive technology and prioritize stakeholder engagement in decision-making.

Genome Editing and NIH Funding

While NIH can't precisely quantify the amount of NIH funding that supported the development of CRISPR given its evolutionary development, it is hundreds of millions of dollars. Genome editing technology is being used widely as a research tool across NIH-funded projects and most NIH Institutes and Centers fund such research.

However, identifying precise levels of funding for a technique with so many uses is difficult. That is because searching the terms, "genome or gene editing," "CRISPR," etc., in NIH RePORTER, a database of NIH-funded research, is comparable to searching a term such as "DNA sequencing." If used as a research tool, some researchers may mention it in their RePORTER entry, while others may not. Also, research that results from a search on the word "CRISPR" does not necessarily mean that CRISPR is used in the research. For example, a researcher may reference CRISPR in the RePORTER summary, but may not be using it in the research.

Images

Video

NIH Director Dr. Francis Collins talks about the importance of somatic cell gene editing and how it could be used to combat genetic diseases. If you would like the files of the video clips, please contact Wole Akinso.

Additional Resources

# data[].attributes.attachment_link.description
Genome editing, also called gene editing, is an area of research seeking to modify genes of living organisms to improve our understanding of gene function and develop ways to use it to treat genetic or acquired diseases.

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Main idea:
The main idea of this article is explain negative points and postivie points
Arguments/facts:
- What Is Gene Editing?
- Why Is Gene Editing Being Studied?
- Is Gene Editing Currently Being Used?
- Is Gene Editing Ethical?

Dialogue:

This is one of the best articles because explain the best points of positive and negative
Summary:
The primary debate around gene editing is whether or not it’s safe to modify human DNA and genetic sequences. Some experts believe that current technology is sufficient, and embryos could greatly benefit from being altered.
Since these consequences are avoidable, they insist that any potential rewards are not worth the risk.
Another common argument stems from the perceived inevitability of non-therapeutic gene editing and enhancement procedures.
APA:
Rex Md (2022, May 18) What Is Gene Editing?
Is It Moral or Ethical? Retrieved from
https://rexmd.com/learn/what-is-gene-editing

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What Is Gene Editing? Is It Moral Or Ethical? | Rex MD

# data[].attributes.attachment_link.description
Gene editing is complicated, and the possibilities are theoretically immense. Is altering human genes ethical? Let’s explore.

# data[].attributes.body
Main idea:
The main idea of this article is that it tells you that in soon the gene editing it would be possible

Arguments/facts:
- In soon it would be possible to do gene editing
-  They discoverd that can be possible diseases
- Taming jumping genes
- Human applications

Dialogue: I didn't now that this topic was so interesant

Summary:
In just 10 years, CRISPR has revolutionized gene editing. The technique has fueled a wave of new scientific discoveries in cancer, Alzheimer’s, and heart disease, and several gene therapies that deploy the gene editor will soon be available to patients.

APA:  Hoffmann . Florian  et al (2022, August 24)The Next Revolution in Gene Editing?
Retrived from : https://www.cuimc.columbia.edu/news/next-revolution-gene-editing

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Columbia University Irving Medical Center

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In just 10 years, CRISPR has revolutionized gene editing. The technique has fueled a wave of new scientific discoveries in cancer, Alzheimer's, and heart disease, and several gene therapies that deploy the gene editor will soon be available to patients.

But the first-generation version of the gene-editing system-CRISPR-Cas9-does have some drawbacks. Though it allows scientists to edit the genome at a precise location, controlling the change that's made is often difficult, and the final edit is hard to predict.

Next-generation gene editors are needed, and a promising new technology, which relies on parasitic "jumping genes," is under development in the laboratory of Sam Sternberg, PhD, assistant professor of biochemistry & molecular biophysics, and may be able to overcome the limitations of the first generation of gene editing.

Taming jumping genes

Jumping genes-also called transposons-are mobile regions in the genome that jump randomly into other parts of the genome. Although transposons often cause harm to the organism due to this lack of specificity, they mobilize using highly efficient "integrase" enzymes that can paste large genetic payloads into the genome. The Sternberg lab previously discovered a unique family of bacterial transposons that naturally exploit CRISPR to control their specificity, and thus offer a new mode of gene editing that has both remarkable accuracy and efficient DNA insertion activity.

Sternberg and his colleagues previously demonstrated that the transposon-based gene editor appears less likely to edit unintended genomic targets than the original CRISPR technology.

One reason the new editor achieves its remarkable accuracy, the researchers report today in the journal Nature, lies in a single protein in the editing complex.

Molecular details of the TnsC protein, including its 3D structure (above), should help the researchers optimize the new gene editor for genome engineering of human cells. Image: Hoffmann, Kim, Beh et al., Nature (2022).

In the new work, Sternberg's team, led by PhD student Florian Hoffmann and postdoctoral scientists Minjoo Kim, PhD, and Leslie Beh, PhD, used biochemical techniques to identify all the sites in the genome that the editor's proteins bind during gene insertion in a bacterium. This approach revealed that while the editor's main protein complex binds many off-target sites, only a small number of those sites can recruit the TnsC protein, which is essential for gene insertion.

The process operates similarly to a set of search filters a user might apply during an advanced search online. "These gene editing systems first sample a large set of possible target sites, and then TnsC is the filter that selects only the correct on-target site for DNA integration," says Sternberg.

The researchers also revealed the 3D structure of TnsC in work led by Israel Fernández, PhD, a collaborator at St. Jude Children's Research Hospital, which revealed that this critical protein forms a ring around the DNA and precisely positions the genomic target site for transposon insertion.

Human applications?

The new details about the molecular mechanics of these transposons as they edit bacterial genomes should help researchers optimize the new gene editors for genome engineering of human cells and clinical applications, Sternberg says.

The transposon-based editor's ability to make large gene insertions with exquisite precision could be especially useful in certain types of gene therapy. While the original CRISPR-Cas9 tool is limited to making small alterations, the new editor could potentially insert complete, functional genes into patient cells.

Sternberg says that although clinical applications such as this will require further technological development, his team has already met one challenge and successfully adapted the gene editor to operate in human cells in the lab.

# data[].attributes.attachment_link.description
In just 10 years, CRISPR has revolutionized gene editing. The technique has fueled a wave of new scientific discoveries in cancer, Alzheimer's, and heart disease, and several gene therapies that deploy the gene editor will soon be available to patients. But the first-generation version of the gene-editing system-CRISPR-Cas9-does have some drawbacks.

# data[].attributes.body
Main idea:
This web page talks basicly about treating desease
Arguments/facts:
-Desease
-Technology
-CRISPR
-Solutions

Dialogue:

How do they treat so clean desease
Summary:
From large-scale screening to focused gene editing, CRISPR is a powerful genome editing technology. Specificity and stability are paramount to the successful implementation of guide RNA , as off-target edits can have significant impacts in downstream applications.
The successful use of DNA oligo pools for CRISPR screening relies upon the fidelity and representation of the starting library.
APA
Advance your research with Agilent CRISPR solutions | Agilent. (n.d.). retrived from:
https://www.agilent.com/en/solutions/genomics-applications-solutions/crispr?ds_rl=1299916

# data[].attributes.attachment_link.keywords[]
agilent sureguide crispr tools and libraries crispr and gene editing chemically modified sgrna

# data[].attributes.attachment_link.title
Advance your research with Agilent CRISPR solutions | Agilent

# data[].attributes.attachment_link.description
From large-scale screening to focused gene editing, CRISPR is a powerful genome editing technology. Drive your research with the broad array of Agilent SureGuide products, ranging from genome-wide and custom CRISPR oligo libraries to highly specific and stable chemically synthesized single guide RNAs (sgRNA).

# data[].attributes.body
Main idea:
It talks about the countrys that are trying to do gene editing.
Arguments/facts:
- Human genome editing: somatic vs. germline
- Bioethics
- Business
- Medicine
- Law
- Science
- Technology, and society

Dialogue: In my opinion I think these is not a good idea because these will give as a lot of problems

Summary: Bassicly they are trying to control as by changing are DNA and win money.

APA
Harvard (January 9, 2019) Perspective of gene editing.

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This article was originally published in the Harvard Gazette on January 9, 2019.
Medicine is at a turning point, on the cusp of major change as disruptive technologies such as gene, RNA, and cell therapies enable scientists to approach diseases in new ways. The swiftness of this change is being driven by innovations such as CRISPR gene editing, which makes it possible to correct errors in DNA with relative ease.

Progress in this field has been so rapid that the dialogue around potential ethical, societal, and safety issues is scrambling to catch up.

This disconnect was brought into stark relief at the Second International Summit on Human Genome Editing, held in Hong Kong in November, when exciting updates about emerging therapies were eclipsed by a disturbing announcement. He Jiankui, a Chinese researcher, claimed that he had edited the genes of two human embryos, and that they had been brought to term.

There was immediate outcry from scientists across the world, and He was subjected to intense social pressure, including the removal of his affiliations, for having allegedly disregarded ethical norms and his patients' safety.

Yet as I. Glenn Cohen '03, faculty director of the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School, has said, gene editing comes in many varieties, with many consequences. Any deep ethical discussion needs to take into account those distinctions.

Human genome editing: somatic vs. germline

The germline editing He claimed to have carried out is quite different from the somatic gene therapies that are currently changing the frontiers of medicine. While somatic gene editing affects only the patient being treated (and only some of his or her cells), germline editing affects all cells in an organism, including eggs and sperm, and so is passed on to future generations. The possible consequences of that are difficult to predict.

Somatic gene therapies involve modifying a patient's DNA to treat or cure a disease caused by a genetic mutation. In one clinical trial, for example, scientists take blood stem cells from a patient, use CRISPR techniques to correct the genetic mutation causing them to produce defective blood cells, then infuse the "corrected" cells back into the patient, where they produce healthy hemoglobin. The treatment changes the patient's blood cells, but not his or her sperm or eggs.

Germline human genome editing, on the other hand, alters the genome of a human embryo at its earliest stages. This may affect every cell, which means it has an impact not only on the person who may result, but possibly on his or her descendants. There are, therefore, substantial restrictions on its use.

Germline editing in a dish can help researchers figure out what the health benefits could be, and how to reduce risks. Those include targeting the wrong gene; off-target impacts, in which editing a gene might fix one problem but cause another; and mosaicism, in which only some copies of the gene are altered. For these and other reasons, the scientific community approaches germline editing with caution, and the U.S. and many other countries have substantial policy and regulatory restrictions on using germline human genome editing in people.

But many scientific leaders are asking: When the benefits are believed to outweigh the risks, and dangers can be avoided, should science consider moving forward with germline genome editing to improve human health? If the answer is yes, how can researchers do so responsibly?

CRISPR pioneer Feng Zhang of the Broad Institute of Harvard and MIT responded immediately to He's November announcement by calling for a moratorium on implanting edited embryos in humans. Later, at a public event on "Altering the Human Genome" at the Belfer Center at Harvard Kennedy School (HKS), he explained why he felt it was important to wait:

"The moratorium is a pause. Society needs to figure out if we all want to do this, if this is good for society, and that takes time. If we do, we need to have guidelines first so that the people who do this work can proceed in a responsible way, with the right oversight and quality controls."

Professors at the University's schools of medicine, law, business, and government saw He's announcement as a turning point in the discussion about heritable gene therapies and shared their perspectives on the future of this technology with the Gazette.

Here are their thoughts, issue by issue:

Bioethics

Aside from the safety risks, human genome editing poses some hefty ethical questions. For families who have watched their children suffer from devastating genetic diseases, the technology offers the hope of editing cruel mutations out of the gene pool. For those living in poverty, it is yet another way for the privileged to vault ahead. One open question is where to draw the line between disease treatment and enhancement, and how to enforce it, considering differing attitudes toward conditions such as deafness.

Robert Truog, director of the Center for Bioethics at Harvard Medical School (HMS), provided context:

"This question is not as new as it seems. Evolution progresses by random mutations in the genome, which dwarf what can be done artificially with CRISPR. These random mutations often cause serious problems, and people are born with serious defects. In addition, we have been manipulating our environment in so many ways and exposing ourselves to a lot of chemicals that cause unknown changes to our genome. If we are concerned about making precise interventions to cure disease, we should also be interested in that.

"To me, the conversation around Dr. He is not about the fundamental merits of germline gene editing, which in the long run will almost certainly be highly beneficial. Instead, it's about the oversight of science. The concern is that with technologies that are relatively easy to use, like CRISPR, how does the scientific community regulate itself? If there's a silver lining to this cloud, I think it is that the scientific community did pull together to be critical of this work, and took the responsibility seriously to use the tools available to them to regulate themselves."

Business

When asked what the implications of He's announcement are for the emerging field of precision medicine, Richard Hamermesh, faculty co-chair of the Harvard Business School/Kraft Precision Medicine Accelerator, said:

"Before we start working on embryos, we have a long way to go, and civilization has to think long and hard about it. There's no question that gene editing technologies are potentially transformative and are the ultimate precision medicine. If you could precisely correct or delete genes that are causing problems-mutating or aberrant genes-that is the ultimate in precision. It would be so transformative for people with diseases caused by a single gene mutation, like sickle cell anemia and cystic fibrosis. Developing safe, effective ways to use gene editing to treat people with serious diseases with no known cures has so much potential to relieve suffering that it is hard to see how anyone could be against it.

"There is also commercial potential and that will drive it forward. A lot of companies are getting venture funding for interesting gene therapies, but they're all going after tough medical conditions where there is an unmet need-[where] nothing is working-and they're trying to find gene therapies to cure those diseases. Why should we stop trying to find cures?

"But anything where you're going to be changing human embryos, it's going to take a long time for us to figure out what is appropriate and what isn't. That has to be done with great care in terms of ethics."

Medicine

George Q. Daley is dean of HMS, the Caroline Shields Walker Professor of Medicine, and a leader in stem cell science and cancer biology. As a spokesperson for the organizing committee of the Second International Summit on Human Genome Editing, he responded swiftly to He's announcement in Hong Kong. Echoing those remarks, he said:

"It's time to formulate what a clinical path to translation might look like so that we can talk about it. That does not mean that we're ready to go into the clinic-we are not. We need to specify what the hurdles would be if one were to move forward responsibly and ethically. If you can't surmount those hurdles, you don't move forward.

"There are stark distinctions between editing genes in an embryo to prevent a baby from being born with sickle cell anemia and editing genes to alter the appearance or intelligence of future generations. There is a whole spectrum of considerations to be debated. The prospect includes an ultimate decision that we not go forward, that we decide that the benefits do not outweigh the costs."

Asked how to prevent experiments like He's while preserving academic freedom, Daley replied:

"For the past 15 years, I have been involved in efforts to establish international standards of professional conduct for stem cell research and its clinical translation, knowing full well that there could be-and has been-a growing number of independent practitioners directly marketing unproven interventions to vulnerable patients through the internet. We advocated so strongly for professional standards in an attempt to ward off the risks of an unregulated industry. Though imperfect, our efforts to encourage a common set of professional practices have been influential.

"You can't control rogue scientists in any field. But with strongly defined guidelines for responsible professional conduct in place, such ethical violations like those of Dr. He should remain a backwater, because most practitioners will adhere to generally accepted norms. Scientists have a responsibility to come together to articulate professional standards and live by them. One has to raise the bar very high to define what the standards of safety and efficacy are, and what kind of oversight and independent judgment would be required for any approval.

"We have called for an ongoing international forum on human genome editing, and that could take many shapes. We've suggested that the national academies of more countries come together-the National Academy of Sciences in the U.S. and the Royal Society in the U.K. are very active here-because these are the groups most likely to have the expertise to convene these kinds of discussions and keep them going."

Law

Cohen, speaking to the legal consequences of germline human genome editing, said:

"I think we should slow down in our reaction to this case. It is not clear that the U.S. needs to react to Dr. He's announcement with regulation. The FDA [Food and Drug Administration] already has a strong policy on germline gene editing in place. A rider in the Consolidated Appropriations Act of 2016-since renewed-would have blocked the very same clinical application of human germline editing He announced, had it been attempted in the U.S.

"The scientific community has responded in the way I'd have liked it to. There is a difference between 'governance' and 'self-governance.' Where government uses law, the scientific community uses peer review, public censure, promotions, university affiliations, and funding to regulate themselves. In China, in Dr. He's case, you have someone who's (allegedly) broken national law and scientific conventions. That doesn't mean you should halt research being done by everyone who's law-abiding.

"Public policy or ethical discussion that's divorced from how science is progressing is problematic. You need to bring everyone together to have robust discussions. I'm optimistic that this is happening, and has happened. It's very hard to deal with a transnational problem with national legislation, but it would be great to reach international consensus on this subject. These efforts might not succeed, but ultimately they are worth pursuing."

Science

Professor Kevin Eggan of Harvard's Department of Stem Cell and Regenerative Biology said, "The question we should focus on is: Will this be safe and help the health of a child? Can we demonstrate that we can fix a mutation that will cause a terrible health problem, accurately and without the risk of harming their potential child? If the answer is yes, then I believe germline human genome editing is likely to gain acceptance in time.

"There could be situations where it could help a couple, but the risks of something going wrong are real. But at this point, it would be impossible to make a risk-benefit calculation in a responsible manner for that couple. Before we could ever move toward the clinic, the scientific community must come to a consensus on how to measure success, and how to measure off-target effects in animal models.

"Even as recently as this past spring and fall, the results of animal studies using CRISPR-the same techniques Dr. He claimed to have used-generated a lot of confusion. There is disagreement about both the quality of the data and how to interpret it. Until we can come to agreement about what the results of animal experiments mean, how could we possibly move forward with people?

"As happened in England with mitochondrial replacement therapy, we should be able to come to both a scientific and a societal consensus of when and how this approach should be used. That's missing."

According to Catherine Racowsky, professor of obstetrics, gynecology and reproductive biology at Brigham and Women's Hospital, constraints on the use of embryos in federally funded research pose barriers to studying the risks and benefits of germline editing in humans. She added:

"Until the work is done, carefully and with tight oversight, to understand any off-target effects of replacing or removing a particular gene, it is inappropriate to apply the technology in the clinical field. My understanding of Dr. He's case is that there wasn't a known condition in these embryos, and by editing the genes involved with HIV infection, he could also have increased the risks of susceptibility to influenza and West Nile viruses.

"We need a sound oversight framework, and it needs to be established globally. This is a technology that holds enormous promise, and it is likely to be applied to the embryo, but it should only be applied for clinical purposes after the right work has been done. That means we must have consensus on what applications are acceptable, that we have appropriate regulatory oversight, and, perhaps most importantly, that it is safe. The only way we're going to be able to determine that these standards are met is to proceed cautiously, with reassessments of the societal and health benefits and the risks."

Asked about public dialogue around germline human genome editing, George Church, Robert Winthrop Professor of Genetics at HMS, said:

"With in vitro fertilization (IVF), 'test tube babies' was an intentionally scary term. But after Louise Brown, the first IVF baby, was born healthy 40 years ago, attitudes changed radically. Ethics flipped 180 degrees, from it being a horrifying idea to being unacceptable to prevent parents from having children by this new method. If these edited twins are proven healthy, very different discussions will arise. For example, is a rate of 900,000 deaths from HIV infection per year a greater risk than West Nile virus, or influenza? How effective is each vaccine?"

Science, technology, and society

Sheila Jasanoff, founding director of the Science, Technology, and Society program at HKS, has been calling for a "global observatory" on gene editing, an international network of scholars and organizations dedicated to promoting exchange across disciplinary and cultural divides. She said:

"The notion that the only thing we should care about is the risk to individuals is very American. So far, the debate has been fixated on potential physical harm to individuals, and not anything else. This is not a formulation shared with other countries in the world, including practically all of Europe. Considerations of risk have equally to do with societal risk. That includes the notion of the family, and what it means to have a 'designer baby.'

"These were not diseased babies Dr. He was trying to cure. The motivation for the intervention was that they live in a country with a high stigma attached to HIV/AIDS, and the father had it and agreed to the intervention because he wanted to keep his children from contracting AIDS. AIDS shaming is a fact of life in China, and now it won't be applied to these children. So, are we going to decide that it's OK to edit as-yet-to-be children to cater to this particular idea of a society?

"It's been said that 'the genie is out of the bottle' with germline human genome editing. I just don't think that's true. After all, we have succeeded in keeping 'nuclear' inside the bottle. Humanity doesn't lack the will, intelligence, or creativity to come up with ways for using technology for good and not ill.

"We don't require students to learn the moral dimensions of science and technology, and that has to change. I think we face similar challenges in robotics, artificial intelligence, and all kinds of frontier fields that have the potential to change not just individuals but the entirety of what it means to be a human being.

"Science has this huge advantage over most professional thought in that it has a universal language. Scientists can hop from lab to lab internationally in a way that lawyers cannot because laws are written in many languages and don't translate easily. It takes a very long time for people to understand each other across these boundaries. A foundational concept for human dignity? It would not be the same thing between cultures.

"I would like to see a 'global observatory' that goes beyond gene editing and addresses emerging technologies more broadly."
To learn more: Petrie-Flom Center Annual Conference: Consuming Genetics: Ethical and Legal Considerations of New Technologies: http://petrieflom.law.harvard.edu/events/details/2019-petrie-flom-center-annual-conference

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Perspectives on gene editing - Harvard Law School

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This article was originally published in the Harvard Gazette on January 9, 2019. Medicine is at a turning point, on the cusp of major change as disruptive technologies such as gene, RNA, and cell therapies enable scientists to approach diseases in new ways.

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Main idea:
The main idea is to control the variant of covid(omicron variant)
Arguments/facts:
-Covid
-Omicron
-Gene editing
-Dna

Dialogue:

Why theres so many variants of covid 19?
Summary:
CrisprBits, one of the first Indian companies using CRISPR, a breakthrough gene-editing technology, on Tuesday announced that it has developed OmiCrisp, a CRISPR- based test to detect SARS-CoV2. The comany claims that the test can also determine whether it is an Omicron or non-omicron variant. « The patent pending OmiCrisp test uses the Cas12a enzyme and separate guides to target variant-specific sequences found in Omicron and non-Omicron variants of SARS-CoV-2,» company stated on Tuesday. «The test was tested on over 80 clinical samples and was 100 pecent accurate in identifying whether a sample was Omicron or non-Omicron when compared to sequencing results.
APA
Health desk(January 24)CrisprBits develops gene-editing technology-based test to detect Omicron variant
retrived from:
https://www.financialexpress.com/healthcare/covid-19/crisprbits-develops-gene-editing-technology-based-test-to-detect-omicron-variant/2958618/

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CrisprBits develops gene-editing technology-based test to detect Omicron variant

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The company also claimed that this is different from many other tests in the market that use RT-PCR signal drop-out to identify Omicron variants.

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Main idea:
What is Gene Editing
Arguments/facts:
- A Delicate Editing Tool for DNA
- Gene editing: A biomedical “swiss army knife”
- From Research Lab to Real World Use

Dialogue:

Making special tools for gene editing
Summary:
One groundbreaking area of focus is gene editing, a technique that allows scientists to, for example, correct faulty genes that cause a specific disease, thereby aiming to reverse certain disease symptoms or prevent the disease from occurring in the first place.
APA
Unknown (2020,5 noviembre) What is gene editing  https://www.bayer.com/en/news-stories/what-is-gene-editing

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Gene editing encompasses many different tools and technologies, but the end-goal is the same: to address the root cause of a disease instead of managing its outcome, i.e., the symptoms

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Main idea:
Good things of gene editing
Arguments/facts:
- Idustry
- Guide
- Purification
- Method

Dialogue: I think these is a better way of to try to tell us that gene editing is good and in these point of view gene editing is good.

Summary: Every guide, including the target and tracrRNA sequence, is produced using fully customized de novo synthesis.

APA
Unknown  retrevied (31/1/2023) Advance your research with Agilent gene editing solutions

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Advance your research with Agilent CRISPR solutions | Agilent

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From large-scale screening to focused gene editing, CRISPR is a powerful genome editing technology. Drive your research with the broad array of Agilent SureGuide products, ranging from genome-wide and custom CRISPR oligo libraries to highly specific and stable chemically synthesized single guide RNA…

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Main idea:
It talks about the changes of gene editing
Arguments/facts:
- Early attempts to correct genetic mistakes
- Breaking DNA at desired locations
- Applications and controversies
- three-parent baby

Dialogue:

This article is not very essy to understand but, when you understand is a very good article.

Summary:
Gene editing is performed using enzymes, particularly nucleases that have been engineered to target a specific DNA sequence, where they introduce cuts into the DNA strands, enabling the removal of existing DNA and the insertion of replacement DNA. Key among gene-editing technologies is a molecular tool known as CRISPR-Cas9, a powerful technology discovered in 2012 by American scientist Jennifer Doudna, French scientist Emmanuelle Charpentier, and colleagues and refined by American scientist Feng Zhang and colleagues.

APA: Rogers,Kara . Fridovichfeli,Judith (2016, January 07)
Britannica
retrived from: https://www.britannica.com/science/gene-editing

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Table of Contents

gene editing, the ability to make highly specific changes in the DNA sequence of a living organism, essentially customizing its genetic makeup. Gene editing is performed using enzymes, particularly nucleases that have been engineered to target a specific DNA sequence, where they introduce cuts into the DNA strands, enabling the removal of existing DNA and the insertion of replacement DNA. Key among gene-editing technologies is a molecular tool known as CRISPR-Cas9, a powerful technology discovered in 2012 by American scientist Jennifer Doudna, French scientist Emmanuelle Charpentier, and colleagues and refined by American scientist Feng Zhang and colleagues. CRISPR-Cas9 functioned with precision, allowing researchers to remove and insert DNA in the desired locations.

The significant leap in gene-editing tools brought new urgency to long-standing discussions about the ethical and social implications surrounding the genetic engineering of humans. Many questions, such as whether genetic engineering should be used to treat human disease or to alter traits such as beauty or intelligence, had been asked in one form or another for decades. With the introduction of facile and efficient gene-editing technologies, particularly CRISPR-Cas9, however, those questions were no longer theoretical, and the answers to them stood to have very real impacts on medicine and society.

Early attempts to correct genetic mistakes

The idea of using gene editing to treat disease or alter traits dates to at least the 1950s and the discovery of the double-helix structure of DNA. In the mid-20th-century era of genetic discovery, researchers realized that the sequence of bases in DNA is passed (mostly) faithfully from parent to offspring and that small changes in the sequence can mean the difference between health and disease. Recognition of the latter led to the inescapable conjecture that with the identification of "molecular mistakes" that cause genetic diseases would come the means to fix those mistakes and thereby enable the prevention or reversal of disease. That notion was the fundamental idea behind gene therapy and from the 1980s was seen as a holy grail in molecular genetics.

The development of gene-editing technology for gene therapy, however, proved difficult. Much early progress focused not on correcting genetic mistakes in the DNA but rather on attempting to minimize their consequence by providing a functional copy of the mutated gene, either inserted into the genome or maintained as an extrachromosomal unit (outside the genome). While that approach was effective for some conditions, it was complicated and limited in scope.

In order to truly correct genetic mistakes, researchers needed to be able to create a double-stranded break in DNA at precisely the desired location in the more than three billion base pairs that constitute the human genome. Once created, the double-stranded break could be efficiently repaired by the cell using a template that directed replacement of the "bad" sequence with the "good" sequence. However, making the initial break at precisely the desired location-and nowhere else-within the genome was not easy.

Breaking DNA at desired locations

Before the advent of CRISPR-Cas9, two approaches were used to make site-specific double-stranded breaks in DNA: one based on zinc finger nucleases (ZFNs) and the other based on transcription activator-like effector nucleases (TALENs). ZFNs are fusion proteins composed of DNA-binding domains that recognize and bind to specific three- to four-base-pair-long sequences. Conferring specificity to a nine-base-pair target sequence, for example, would require three ZFN domains fused in tandem. The desired arrangement of DNA-binding domains is also fused to a sequence that encodes one subunit of the bacterial nuclease Fok1. Facilitating a double-stranded cut at a specific site requires the engineering of two ZFN fusion proteins-one to bind on each side of the target site, on opposite DNA strands. When both ZFNs are bound, the Fok1 subunits, being in proximity, bind to each other to form an active dimer that cuts the target DNA on both strands.

TALEN fusion proteins are designed to bind to specific DNA sequences that flank a target site. But instead of using zinc finger domains, TALENs utilize DNA-binding domains derived from proteins from a group of plant pathogens. For technical reasons TALENs are easier to engineer than ZFNs, especially for longer recognition sites. Similar to ZFNs, TALENs encode a Fok1 domain fused to the engineered DNA-binding region, so, once the target site is bound on both sides, the dimerized Fok1 nuclease can introduce a double-stranded break at the desired DNA location.

Unlike ZFNs and TALENs, CRISPR-Cas9 uses RNA-DNA binding, rather than protein-DNA binding, to guide nuclease activity, which simplifies the design and enables application to a broad range of target sequences. CRISPR-Cas9 was derived from the adaptive immune systems of bacteria. The acronym CRISPR refers to clustered regularly interspaced short palindromic r epeats, which are found in most bacterial genomes. Between the short palindromic repeats are stretches of sequence clearly derived from the genomes of bacterial pathogens. "Older" spacers are found at the distal end of the cluster, and "newer" spacers, representing more recently encountered pathogens, are found near the proximal end of the cluster.

Transcription of the CRISPR region results in the production of small "guide RNAs" that include hairpin formations from the palindromic repeats linked to sequences derived from the spacers, allowing each to attach to its corresponding target. The RNA-DNA heteroduplex formed then binds to a nuclease called Cas9 and directs it to catalyze the cleavage of double-stranded DNA at a position near the junction of the target-specific sequence and the palindromic repeat in the guide RNA. Because RNA-DNA heteroduplexes are stable and because designing an RNA sequence that binds specifically to a unique target DNA sequence requires only knowledge of the Watson-Crick base-pairing rules (adenine binds to thymine [or uracil in RNA], and cytosine binds to guanine), the CRISPR-Cas9 system was preferable to the fusion protein designs required for using ZFNs or TALENs.

A further technical advance came in 2015, when Zhang and colleagues reported the application of Cpf-1, rather than Cas9, as the nuclease paired with CRISPR to achieve gene editing. Cpf-1 is a microbial nuclease that offers potential advantages over Cas9, including requiring only one CRISPR guide RNA for specificity and making staggered (rather than blunt) double-stranded DNA cuts. The altered nuclease properties gave potentially greater control over the insertion of replacement DNA sequences than was possible with Cas9, at least in some circumstances. Researchers suspect that bacteria house other genome-editing proteins as well, the evolutionary diversity of which could prove valuable in further refining the precision and versatility of gene-editing technologies.

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Gene editing | Definition, History, & CRISPR-Cas9

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gene editing, the ability to make highly specific changes in the DNA sequence of a living organism, essentially customizing its genetic makeup. Gene editing is performed using enzymes, particularly nucleases that have been engineered to target a specific DNA sequence, where they introduce cuts into the DNA strands, enabling the removal of existing DNA and the insertion of replacement DNA.

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Main idea:
tooks about the gene editing
Arguments/facts:
- History
- Background
- Gene targeting

- Process

Dialogue:

I thing that is a good web or article but there are better articles of this topic because they don't explain a lot of thing, but a good thing that they do is that explains the history
Summary:
Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain , double-strand breaks in target DNA by the restriction endonucleases , and the repair of DSBs through homology-directed recombination or non-homologous end joining .
APA :
Wikipedia contributors,(2022, december, 11) Genome editing
retrived form: https://en.wikipedia.org/wiki/Genome_editing

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Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases ( FokI and Cas), and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

Genome editing was pioneered in the 1990s, before the advent of the common current nuclease-based gene editing platforms but its use was limited by low efficiencies of editing. Genome editing with engineered nucleases, i.e. all three major classes of these enzymes-zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered meganucleases-were selected by Nature Methods as the 2011 Method of the Year. The CRISPR-Cas system was selected by as 2015 Breakthrough of the Year.

As of 2015 four families of engineered nucleases were used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats ( CRISPR/ Cas9) system. Nine genome editors were available as of 2017.

In 2018, the common methods for such editing used engineered nucleases, or "molecular scissors". These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits').

In May 2019, lawyers in China reported, in light of the purported creation by Chinese scientist He Jiankui of the first gene-edited humans (see Lulu and Nana controversy), the drafting of regulations that anyone manipulating the human genome by gene-editing techniques, like CRISPR, would be held responsible for any related adverse consequences. A cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies has been recently discussed, focusing on the stochastic nature of cellular control processes.

The University of Edinburgh Roslin Institute engineered pigs resistant to a virus that causes porcine reproductive and respiratory syndrome, which costs US and European pig farmers $2.6 billion annually.

In 2021, England (not the rest of the UK) planned to remove restrictions on gene-edited plants and animals, moving from European Union-compliant regulation to rules closer to those of the US and some other countries. An April 2021 European Commission report found "strong indications" that the current regulatory regime was not appropriate for gene editing Later in 2021, researchers announced a CRISPR alternative, labeled obligate mobile element-guided activity (OMEGA) proteins including IscB, IsrB and TnpB as endonucleases found in transposons, and guided by small ωRNAs.

Genetic engineering as method of introducing new genetic elements into organisms has been around since the 1970s. One drawback of this technology has been the random nature with which the DNA is inserted into the hosts genome, which can impair or alter other genes within the organism. Although, several methods have been discovered which target the inserted genes to specific sites within an organism genome. It has also enabled the editing of specific sequences within a genome as well as reduced off target effects. This could be used for research purposes, by targeting mutations to specific genes, and in gene therapy. By inserting a functional gene into an organism and targeting it to replace the defective one it could be possible to cure certain genetic diseases.

Early methods to target genes to certain sites within a genome of an organism (called gene targeting) relied on homologous recombination (HR). By creating DNA constructs that contain a template that matches the targeted genome sequence it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on embryonic stem cells led to the development of transgenic mice with targeted genes knocked out. It has also been possible to knock in genes or alter gene expression patterns. In recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine.

If a vital gene is knocked out it can prove lethal to the organism. In order to study the function of these genes site specific recombinases (SSR) were used. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognising FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that express the SSR under control of tissue specific promoters, it is possible to knock out or switch on genes only in certain cells. These techniques were also used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or stages of development.

dsDNA-break repair pathways and genome editing using CRISPR-Cas nucleases

A common form of Genome editing relies on the concept of DNA double stranded break (DSB) repair mechanics. There are two major pathways that repair DSB; non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ uses a variety of enzymes to directly join the DNA ends while the more accurate HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point. This can be exploited by creating a vector with the desired genetic elements within a sequence that is homologous to the flanking sequences of a DSB. This will result in the desired change being inserted at the site of the DSB. While HDR based gene editing is similar to the homologous recombination based gene targeting, the rate of recombination is increased by at least three orders of magnitude.

The key to genome editing is creating a DSB at a specific point within the genome. Commonly used restriction enzymes are effective at cutting DNA, but generally recognize and cut at multiple sites. To overcome this challenge and create site-specific DSB, three distinct classes of nucleases have been discovered and bioengineered to date. These are the Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases ( TALEN), meganucleases and the clustered regularly interspaced short palindromic repeats ( CRISPR/Cas9) system.

Meganucleases, discovered in the late 1980s, are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence.

Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific. However, there is virtually no chance of finding the exact meganuclease required to act on a chosen specific DNA sequence. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence. Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucelases in a method named rationally designed meganuclease. Another approach involves using computer models to try to predict as accurately as possible the activity of the modified meganucleases and the specificity of the recognized nucleic sequence.

A large bank containing several tens of thousands of protein units has been created. These units can be combined to obtain chimeric meganucleases that recognize the target site, thereby providing research and development tools that meet a wide range of needs (fundamental research, health, agriculture, industry, energy, etc.) These include the industrial-scale production of two meganucleases able to cleave the human XPC gene; mutations in this gene result in Xeroderma pigmentosum, a severe monogenic disorder that predisposes the patients to skin cancer and burns whenever their skin is exposed to UV rays.

Zinc finger motifs occur in several transcription factors. The zinc ion, found in 8% of all human proteins, plays an important role in the organization of their three-dimensional structure. In transcription factors, it is most often located at the protein-DNA interaction sites, where it stabilizes the motif. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence.

The method generally adopted for this involves associating two DNA binding proteins - each containing 3 to 6 specifically chosen zinc fingers - with the catalytic domain of the FokI endonuclease which need to dimerize to cleave the double-strand DNA. The two proteins recognize two DNA sequences that are a few nucleotides apart. Linking the two zinc finger proteins to their respective sequences brings the two FokI domains closer together. FokI requires dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner would recognize a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered that can only function as heterodimers.

Zinc finger nucleases are research and development tools that have already been used to modify a range of genomes, in particular by the laboratories in the Zinc Finger Consortium. The US company Sangamo BioSciences uses zinc finger nucleases to carry out research into the genetic engineering of stem cells and the modification of immune cells for therapeutic purposes. Modified T lymphocytes are currently undergoing phase I clinical trials to treat a type of brain tumor ( glioblastoma) and in the fight against AIDS.

Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins that feature an array of 33 or 34-amino acid repeats. TALENs are artificial restriction enzymes designed by fusing the DNA cutting domain of a nuclease to TALE domains, which can be tailored to specifically recognize a unique DNA sequence. These fusion proteins serve as readily targetable "DNA scissors" for gene editing applications that enable to perform targeted genome modifications such as sequence insertion, deletion, repair and replacement in living cells. The DNA binding domains, which can be designed to bind any desired DNA sequence, comes from TAL effectors, DNA-binding proteins excreted by plant pathogenic Xanthomanos app. TAL effectors consists of repeated domains, each of which contains a highly conserved sequence of 34 amino acids, and recognize a single DNA nucleotide within the target site. The nuclease can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions. Each repeat is conserved, with the exception of the so-called repeat variable di-residues (RVDs) at amino acid positions 12 and 13. The RVDs determine the DNA sequence to which the TALE will bind. This simple one-to-one correspondence between the TALE repeats and the corresponding DNA sequence makes the process of assembling repeat arrays to recognize novel DNA sequences straightforward. These TALEs can be fused to the catalytic domain from a DNA nuclease, FokI, to generate a transcription activator-like effector nuclease (TALEN). The resultant TALEN constructs combine specificity and activity, effectively generating engineered sequence-specific nucleases that bind and cleave DNA sequences only at pre-selected sites. The TALEN target recognition system is based on an easy-to-predict code. TAL nucleases are specific to their target due in part to the length of their 30+ base pairs binding site. TALEN can be performed within a 6 base pairs range of any single nucleotide in the entire genome.

TALEN constructs are used in a similar way to designed zinc finger nucleases, and have three advantages in targeted mutagenesis: (1) DNA binding specificity is higher, (2) off-target effects are lower, and (3) construction of DNA-binding domains is easier.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as a kind of acquired immunity to protect against viruses. They consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences. By introducing plasmids containing Cas genes and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position.

Editing by nucleobase modification (Base editing)

One of the earliest methods of efficiently editing nucleic acids employs nucleobase modifying enzymes directed by nucleic acid guide sequences was first described in the 1990s and has seen resurgence more recently. This method has the advantage that it does not require breaking the genomic DNA strands, and thus avoids the random insertion and deletions associated with DNA strand breakage. It is only appropriate for precise editing requiring single nucleotide changes and has found to be highly efficient for this type of editing.

ARCUT stands for artificial restriction DNA cutter, it is a technique developed by Komiyama. This method uses pseudo-complementary peptide nucleic acid (pcPNA), for identifying cleavage site within the chromosome. Once pcPNA specifies the site, excision is carried out by cerium (CE) and EDTA (chemical mixture), which performs the splicing function.

TALE nucleases being the most precise and specific method yields a higher efficiency than the previous two methods. It achieves such efficiency because the DNA-binding element consists of an array of TALE subunits, each of them having the capability of recognizing a specific DNA nucleotide chain independent from others, resulting in a higher number of target sites with high precision. New TALE nucleases take about one week and a few hundred dollars to create, with specific expertise in molecular biology and protein engineering.

Because off-target activity of an active nuclease would have potentially dangerous consequences at the genetic and organismal levels, the precision of meganucleases, ZFNs, CRISPR, and TALEN-based fusions has been an active area of research. While variable figures have been reported, ZFNs tend to have more cytotoxicity than TALEN methods or RNA-guided nucleases, while TALEN and RNA-guided approaches tend to have the greatest efficiency and fewer off-target effects. Based on the maximum theoretical distance between DNA binding and nuclease activity, TALEN approaches result in the greatest precision.

Multiplex Automated Genomic Engineering (MAGE)

The methods for scientists and researchers wanting to study genomic diversity and all possible associated phenotypes were very slow, expensive, and inefficient. Prior to this new revolution, researchers would have to do single-gene manipulations and tweak the genome one little section at a time, observe the phenotype, and start the process over with a different single-gene manipulation. Therefore, researchers at the Wyss Institute at Harvard University designed the MAGE, a powerful technology that improves the process of in vivo genome editing. It allows for quick and efficient manipulations of a genome, all happening in a machine small enough to put on top of a small kitchen table. Those mutations combine with the variation that naturally occurs during cell mitosis creating billions of cellular mutations.

MAGE experiments can be divided into three classes, characterized by varying degrees of scale and complexity: (i) many target sites, single genetic mutations; (ii) single target site, many genetic mutations; and (iii) many target sites, many genetic mutations. An example of class three was reflected in 2009, where Church and colleagues were able to program Escherichia coli to produce five times the normal amount of lycopene, an antioxidant normally found in tomato seeds and linked to anti-cancer properties. They applied MAGE to optimize the 1-deoxy-D-xylulose 5-phosphate (DXP) metabolic pathway in Escherichia coli to overproduce isoprenoid lycopene. It took them about 3 days and just over $1,000 in materials. The ease, speed, and cost efficiency in which MAGE can alter genomes can transform how industries approach the manufacturing and production of important compounds in the bioengineering, bioenergy, biomedical engineering, synthetic biology, pharmaceutical, agricultural, and chemical industries.

As of 2012 efficient genome editing had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and was becoming a standard experimental strategy in research labs. The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants and the improvements in TALEN-based approaches testify to the significance of the methods, and the list is expanding rapidly. Genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove genomic elements and therefore create complex systems. In addition, gene functions can be studied using stem cells with engineered nucleases.

Listed below are some specific tasks this method can carry out:

The combination of recent discoveries in genetic engineering, particularly gene editing and the latest improvement in bovine reproduction technologies (e.g. in vitro embryo culture) allows for genome editing directly in fertilised oocytes using synthetic highly specific endonucleases. RNA-guided endonucleases:clustered regularly interspaced short palindromic repeats associated Cas9 (CRISPR/Cas9) are a new tool, further increasing the range of methods available. In particular CRISPR/Cas9 engineered endonucleases allows the use of multiple guide RNAs for simultaneous Knockouts (KO) in one step by cytoplasmic direct injection (CDI) on mammalian zygotes.

Furthermore, gene editing can be applied to certain types of fish in aquaculture such as Atlantic salmon. Gene editing in fish is currently experimental, but the possibilities include growth, disease resistance, sterility, controlled reproduction, and colour. Selecting for these traits can allow for a more sustainable environment and better welfare for the fish.

Thanks to the parallel development of single-cell transcriptomics, genome editing and new stem cell models we are now entering a scientifically exciting period where functional genetics is no longer restricted to animal models but can be performed directly in human samples. Single-cell gene expression analysis has resolved a transcriptional road-map of human development from which key candidate genes are being identified for functional studies. Using global transcriptomics data to guide experimentation, the CRISPR based genome editing tool has made it feasible to disrupt or remove key genes in order to elucidate function in a human setting.

Genome editing using Meganuclease, ZFNs, and TALEN provides a new strategy for genetic manipulation in plants and are likely to assist in the engineering of desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes. Progress in such cases have been recently reported in Arabidopsis thaliana and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations. In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case. Such genome modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation. A potentially successful example of the application of genome editing techniques in crop improvement can be found in banana, where scientists used CRISPR/Cas9 editing to inactivate the endogenous banana streak virus in the B genome of banana (Musa spp.) to overcome a major challenge in banana breeding.

In addition, TALEN-based genome engineering has been extensively tested and optimized for use in plants. TALEN fusions have also been used by a U.S. food ingredient company, Calyxt, to improve the quality of soybean oil products and to increase the storage potential of potatoes

Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting. There is a need for reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.

A common delivery method for CRISPR/Cas9 in plants is Agrobacterium-based transformation. T-DNA is introduced directly into the plant genome by a T4SS mechanism. Cas9 and gRNA-based expression cassettes are turned into Ti plasmids, which are transformed in Agrobacterium for plant application. To improve Cas9 delivery in live plants, viruses are being used more effective transgene delivery.

The ideal gene therapy practice is that which replaces the defective gene with a normal allele at its natural location. This is advantageous over a virally delivered gene as there is no need to include the full coding sequences and regulatory sequences when only a small proportions of the gene needs to be altered as is often the case. The expression of the partially replaced genes is also more consistent with normal cell biology than full genes that are carried by viral vectors.

The first clinical use of TALEN-based genome editing was in the treatment of CD19+ acute lymphoblastic leukemia in an 11-month old child in 2015. Modified donor T cells were engineered to attack the leukemia cells, to be resistant to Alemtuzumab, and to evade detection by the host immune system after introduction.

Extensive research has been done in cells and animals using CRISPR-Cas9 to attempt to correct genetic mutations which cause genetic diseases such as Down syndrome, spina bifida, anencephaly, and Turner and Klinefelter syndromes.

In February 2019, medical scientists working with Sangamo Therapeutics, headquartered in Richmond, California, announced the first ever "in body" human gene editing therapy to permanently alter DNA - in a patient with Hunter syndrome. Clinical trials by Sangamo involving gene editing using Zinc Finger Nuclease (ZFN) are ongoing.

Researchers have used CRISPR-Cas9 gene drives to modify genes associated with sterility in A. gambiae, the vector for malaria. This technique has further implications in eradicating other vector borne diseases such as yellow fever, dengue, and Zika.

The CRISPR-Cas9 system can be programmed to modulate the population of any bacterial species by targeting clinical genotypes or epidemiological isolates. It can selectively enable the beneficial bacterial species over the harmful ones by eliminating pathogen, which gives it an advantage over broad-spectrum antibiotics.

Antiviral applications for therapies targeting human viruses such as HIV, herpes, and hepatitis B virus are under research. CRISPR can be used to target the virus or the host to disrupt genes encoding the virus cell-surface receptor proteins. In November 2018, He Jiankui announced that he had edited two human embryos, to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 ( mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.

In January 2019, scientists in China reported the creation of five identical cloned gene-edited monkeys, using the same cloning technique that was used with Zhong Zhong and Hua Hua - the first ever cloned monkeys - and Dolly the sheep, and the same gene-editing Crispr- Cas9 technique allegedly used by He Jiankui in creating the first ever gene-modified human babies Lulu and Nana. The monkey clones were made in order to study several medical diseases.

Because of the ease of use and cost-efficiency of CRISPR, extensive research is currently being done on it. There are now more publications on CRISPR than ZFN and TALEN despite how recent the discovery of CRISPR is. Both CRISPR and TALEN are favored to be the choices to be implemented in large-scale productions due to their precision and efficiency.

Genome editing occurs also as a natural process without artificial genetic engineering. The agents that are competent to edit genetic codes are viruses or subviral RNA-agents.

Although GEEN has higher efficiency than many other methods in reverse genetics, it is still not highly efficient; in many cases less than half of the treated populations obtain the desired changes. For example, when one is planning to use the cell's NHEJ to create a mutation, the cell's HDR systems will also be at work correcting the DSB with lower mutational rates.

Traditionally, mice have been the most common choice for researchers as a host of a disease model. CRISPR can help bridge the gap between this model and human clinical trials by creating transgenic disease models in larger animals such as pigs, dogs, and non-human primates. Using the CRISPR-Cas9 system, the programmed Cas9 protein and the sgRNA can be directly introduced into fertilized zygotes to achieve the desired gene modifications when creating transgenic models in rodents. This allows bypassing of the usual cell targeting stage in generating transgenic lines, and as a result, it reduces generation time by 90%.

One potential that CRISPR brings with its effectiveness is the application of xenotransplantation. In previous research trials, CRISPR demonstrated the ability to target and eliminate endogenous retroviruses, which reduces the risk of transmitting diseases and reduces immune barriers. Eliminating these problems improves donor organ function, which brings this application closer to a reality.

In plants, genome editing is seen as a viable solution to the conservation of biodiversity. Gene drive are a potential tool to alter the reproductive rate of invasive species, although there are significant associated risks.

Many transhumanists see genome editing as a potential tool for human enhancement. Australian biologist and Professor of Genetics David Andrew Sinclair notes that "the new technologies with genome editing will allow it to be used on individuals (...) to have (...) healthier children" - designer babies. According to a September 2016 report by the Nuffield Council on Bioethics in the future it may be possible to enhance people with genes from other organisms or wholly synthetic genes to for example improve night vision and sense of smell. George Church has compiled a list of potential genetic modifications for possibly advantageous traits such as less need for sleep, cognition-related changes that protect against Alzheimer's disease, disease resistances and enhanced learning abilities along with some of the associated studies and potential negative effects.

The American National Academy of Sciences and National Academy of Medicine issued a report in February 2017 giving qualified support to human genome editing. They recommended that clinical trials for genome editing might one day be permitted once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight."

In the 2016 Worldwide Threat Assessment of the US Intelligence Community statement United States Director of National Intelligence, James R. Clapper, named genome editing as a potential weapon of mass destruction, stating that genome editing conducted by countries with regulatory or ethical standards "different from Western countries" probably increases the risk of the creation of harmful biological agents or products. According to the statement the broad distribution, low cost, and accelerated pace of development of this technology, its deliberate or unintentional misuse might lead to far-reaching economic and national security implications. For instance technologies such as CRISPR could be used to make "killer mosquitoes" that cause plagues that wipe out staple crops.

According to a September 2016 report by the Nuffield Council on Bioethics, the simplicity and low cost of tools to edit the genetic code will allow amateurs - or " biohackers" - to perform their own experiments, posing a potential risk from the release of genetically modified bugs. The review also found that the risks and benefits of modifying a person's genome - and having those changes pass on to future generations - are so complex that they demand urgent ethical scrutiny. Such modifications might have unintended consequences which could harm not only the child, but also their future children, as the altered gene would be in their sperm or eggs. In 2001 Australian researchers Ronald Jackson and Ian Ramshaw were criticized for publishing a paper in the Journal of Virology that explored the potential control of mice, a major pest in Australia, by infecting them with an altered mousepox virus that would cause infertility as the provided sensitive information could lead to the manufacture of biological weapons by potential bioterrorists who might use the knowledge to create vaccine resistant strains of other pox viruses, such as smallpox, that could affect humans. Furthermore, there are additional concerns about the ecological risks of releasing gene drives into wild populations.

In 2020, the Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for "the development of a method for genome editing".

"WHO launches global registry on human genome editing." PharmaBiz, 31 Aug. 2019. Gale General OneFile, Accessed 27 Apr. 2020.

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Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations.

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Main idea:
They make experiments with the gene editing

Arguments/facts:
- Overview
- In the Laboratory
- Treating Disease
- Technical barriers

Dialogue:

This article is very interesant if you like this topic
Summary:
Genome editing tools have the potential to help treat diseases with a genomic basis, like cystic fibrosis and diabetes. This therapy saved Layla's life. However, treatments like the one that Layla received are still experimental because the scientific community and policymakers still have to address technical barriers and ethical concerns surrounding genome editing.
APA:
National human genome (2019, August 15) What is gene editing ?
Retrived from: https://www.genome.gov/about-genomics/policy-issues/what-is-Genome-Editing

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Genome editing technologies enable scientists to make changes to DNA, leading to changes in physical traits, like eye color, and disease risk. Scientists use different technologies to do this. These technologies act like scissors, cutting the DNA at a specific spot. Then scientists can remove, add, or replace the DNA where it was cut.

The first genome editing technologies were developed in the late 1900s. More recently, a new genome editing tool called CRISPR, invented in 2009, has made it easier than ever to edit DNA. CRISPR is simpler, faster, cheaper, and more accurate than older genome editing methods. Many scientists who perform genome editing now use CRISPR.

One way that scientists use genome editing is to investigate different diseases that affect humans. They edit the genomes of animals, like mice and zebrafish, because animals have many of the same genes as humans. For example, mice and humans share about 85 percent of their genes! By changing a single gene or multiple genes in a mouse, scientists can observe how these changes affect the mouse's health and predict how similar changes in human genomes might affect human health.

Scientists at the National Human Genome Research Institute (NHGRI) are doing just this. The Burgess lab, for example, is studying zebrafish genomes. Scientists in this lab delete different genes in zebrafish one at a time using CRISPR to see how the deletion impacts the fish. The Burgess lab focuses on 50 zebrafish genes which are similar to the genes that cause human deafness so that they can better understand the genomic basis of deafness.

In the Laboratory

One way that scientists use genome editing is to investigate different diseases that affect humans. They edit the genomes of animals, like mice and zebrafish, because animals have many of the same genes as humans. For example, mice and humans share about 85 percent of their genes! By changing a single gene or multiple genes in a mouse, scientists can observe how these changes affect the mouse's health and predict how similar changes in human genomes might affect human health.

Scientists at the National Human Genome Research Institute (NHGRI) are doing just this. The Burgess lab, for example, is studying zebrafish genomes. Scientists in this lab delete different genes in zebrafish one at a time using CRISPR to see how the deletion impacts the fish. The Burgess lab focuses on 50 zebrafish genes which are similar to the genes that cause human deafness so that they can better understand the genomic basis of deafness.

Scientists are developing gene therapies - treatments involving genome editing - to prevent and treat diseases in humans. Genome editing tools have the potential to help treat diseases with a genomic basis, like cystic fibrosis and diabetes. There are two different categories of gene therapies: germline therapy and somatic therapy. Germline therapies change DNA in reproductive cells (like sperm and eggs). Changes to the DNA of reproductive cells are passed down from generation to generation. Somatic therapies, on the other hand, target non-reproductive cells, and changes made in these cells affect only the person who receives the gene therapy.

In 2015, scientists successfully used somatic gene therapy when a one-year old in the United Kingdom named Layla received a gene editing treatment to help her fight leukemia, a type of cancer. These scientists did not use CRISPR to treat Layla, and instead used another genome editing technology called TALENs. Doctors tried many treatments before this, but none of them seemed to work, so scientists received special permission to treat Layla using gene therapy. This therapy saved Layla's life. However, treatments like the one that Layla received are still experimental because the scientific community and policymakers still have to address technical barriers and ethical concerns surrounding genome editing.
Technical barriers
Even though CRISPR improved upon older genome editing technologies, it is not perfect. For example, sometimes genome editing tools cut in the wrong spot. Scientists are not yet sure how these errors might affect patients. Assessing the safety of gene therapies and improving upon genome editing technologies are critical steps to ensure that this technology is ready for use in patients.
Ethical concerns
Scientists and all of us should carefully consider the many ethical concerns that can emerge with genome editing, including safety. First and foremost, genome editing must be safe before it is used to treat patients. Some other ethical questions that scientists and society must consider are:

1. Is it okay to use gene therapy on an embryo when it is impossible to get permission from the embryo for treatment? Is getting permission from the parents enough?

2. What if gene therapies are too expensive and only wealthy people can access and afford them? That could worsen existing health inequalities between the rich and poor.

3. Will some people use genome editing for traits not important for health, such as athletic ability or height? Is that okay?

4. Should scientists ever be able to edit germline cells? Edits in the germline would be passed down through generations.

Most people agree that scientists should not edit the genomes of germline cells at this time because the safety and Scientific communities across the world are approaching germline therapy research with caution because edits to a germline cell would be passed down through generations. Many countries and organizations have strict regulations to prevent germline editing for this reason. The NIH, for example, does not fund research to edit human embryos.

Scientists across the world held a conference to talk about these and similar ethical issues at the International Summit on Human Gene Editing.

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Genome editing is a method that lets scientists change the DNA of many organisms, including plants, bacteria, and animals.

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Main idea:
This web page introduce us to gene editing.It tell us what does it mean and it also talks about CRISPR-Cas9(what does it mean.
Arguments/facts:
-Gene editing
-CRISPR
-DNA
-Technology

Dialogue:

I think that this is a better way of changing gene editing
Summary:
Genome editing is a group of technologies that give scientists the ability to change an organism's DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.
Scientists are still working to determine whether this approach is safe and effective for use in people.
APA
What are genome editing and CRISPR-Cas9?: MedlinePlus Genetics. (n.d.). retrived from:
https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/

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Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism's DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A well-known one is called CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other genome editing methods.

CRISPR-Cas9 was adapted from a naturally occurring genome editing system that bacteria use as an immune defense. When infected with viruses, bacteria capture small pieces of the viruses' DNA and insert them into their own DNA in a particular pattern to create segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to "remember" the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays that recognize and attach to specific regions of the viruses' DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

Researchers adapted this immune defense system to edit DNA. They create a small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence in a cell's DNA, much like the RNA segments bacteria produce from the CRISPR array. This guide RNA also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.

Genome editing is of great interest in the prevention and treatment of human diseases. Currently, genome editing is used in cells and animal models in research labs to understand diseases. Scientists are still working to determine whether this approach is safe and effective for use in people. It is being explored in research and clinical trials for a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

Ethical concerns arise when genome editing, using technologies such as CRISPR-Cas9, is used to alter human genomes. Most of the changes introduced with genome editing are limited to somatic cells, which are cells other than egg and sperm cells (germline cells). These changes are isolated to only certain tissues and are not passed from one generation to the next. However, changes made to genes in egg or sperm cells or to the genes of an embryo could be passed to future generations. Germline cell and embryo genome editing bring up a number of ethical challenges, including whether it would be permissible to use this technology to enhance normal human traits (such as height or intelligence). Based on concerns about ethics and safety, germline cell and embryo genome editing are currently illegal in the United States and many other countries.

Scientific journal articles for further reading

Ormond KE(1), Mortlock DP(2), Scholes DT(3), Bombard Y(4), Brody LC(5), Faucett WA(6), Garrison NA(7), Hercher L(8), Isasi R(9), Middleton A(10), Musunuru K(11), Shriner D(12), Virani A(13), Young CE(3). Human Germline Genome Editing. Am J Hum Genet. 2017 Aug 3;101(2):167-176. PubMed: 28777929. Free full-text available from PubMed Central: PMC5544380.

Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest. 2014 Oct;124(10):4154-61. doi: 10.1172/JCI72992. Epub 2014 Oct 1. Review. PubMed: 25271723. Free full-text available from PubMed Central: PMC4191047.

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-78. doi:10.1016/j.cell.2014.05.010. Review. PubMed: 24906146. Free full-text available from PubMed Central: PMC4343198.

Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017 Apr 20;169(3):559. doi:10.1016/j.cell.2017.04.005. PubMed: 28431253.

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What are genome editing and CRISPR-Cas9?: MedlinePlus Genetics

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Gene editing occurs when scientists change the DNA of an organism. Learn more about this process and the different ways it can be done.