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There are few issues which seem to draw stronger reactions both within as well outside of the scientific community than gene cloning or genome editing. They seem to stir something inside many of us about a disruption of the basic components which make up who we are.

A good case in point is CRISPR genome editing, which at this point of its development and application, has had both successful and concerning results. Bringing the issue into even sharper focus, the choice of whether to permit gene editing in the case of human embryos complicates the picture even more.

Securing the support of most countries can be difficult, if not impossible. Perhaps for this reason, the decision by the Japanese government to allow researchers to move forward with the use of gene-editing tools is quite surprising.

The draft guidelines, which were issued by the government on September 28th, will undergo public review next month, and if all goes as expected, will be implemented in 2019.

Although there remain global challenges for conducting research in this area, a string of successful research outcomes were reported in the last year, one of which included the use of gene editing in addressing heart conditions.

Researchers who conduct studies in this controversial area can find loopholes involving private funding or regulatory.

Opinions remain divided on the topic

The critical eyes of the scientific community grew even more critical after a study was published in 2015 by a group of Chinese scientists who genetically modified human embryos, with the results being seen by many as an indication of the global scientific community's need to apply more caution, and wisdom, in approaching this very ethically fragile area of research.

With the help of the CRISPR/Cas9 system, a total of 86 embryos were injected to edit the HBB gene, with 15 not surviving. In addition, among the remaining 71, genetic testing revealed that splicing was successful for only 28.

Junjiu Huang, study leader and gene-function researcher, acknowledged the team's missteps in terms of the study design, stating: "If you want to do it in normal embryos, you need to be close to 100%," adding, "That's why we stopped. We still think it's too immature."

For some others, however, this was a case of too little, too late. "I believe this is the first report of this gene-modifying technique applied to human pre-implantation embryos and as such the study is a landmark, as well as a cautionary tale," George Daley, a stem-cell biologist at Harvard Medical School in Boston, expressed to Nature about the impact of the study.

"Their study should be a stern warning to any practitioner who thinks the technology is ready for testing to eradicate disease genes."

These views together seem to indicate that increased discussionand more clearly defined research parameters are needed across the board to both quiet the critics and those concerned about the ethical implications, and to ensure that scientists continue to do quality work which contributes to the good of humanity. This is hopefully what the Japanese government has in mind with its latest efforts.

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Japan Set to Approve Controversial Gene Editing of Human Embryos

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Japan's draft guidelines for a proposal to begin editing genes found in human embryos was recently announced. The decision has some scientists concerned.

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This article outlines the top 5 most pressing dilemmas facing the world of science in terms of biotechnology.

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Abstract

Biotech healthcare is going through what every other emerging scientific discipline experiences - the challenge of defining its ethical boundaries. Research, costs, and privacy issues spawn concerns that third-party payers, employers, providers, and policy makers will face for years to come.

In an age when medical technology improves with increasing rapidity, the availability of new treatments increases almost as quickly. With advances, however, come dilemmas - scientific, financial, and especially moral. These conundrums are likely to multiply as groups with vastly different viewpoints and resources battle over the direction of health policy.

As the first decade of the 21st century reaches its midpoint, Biotechnology Healthcare has identified five topics that dominate ethical discussions of biotech medicine.1 These issues will continue to generate controversy in the foreseeable future, forcing third-party payers, employer and union purchasers, and health care providers to deal with the policy implications of some or all of them for years.

1. Protecting Human Subjects in Clinical Trials

This issue has generated considerable debate since 1999, when 18-year-old Jesse Gelsinger died while participating in a gene therapy trial at the University of Pennsylvania. The institution was widely criticized for failing to disclose crucial information on informed consent documents, relaxing criteria for accepting volunteers, and enrolling volunteers who were ineligible. The episode prompted a great deal of soul-searching among researchers and regulators, and many universities began implementing new standards as a result of the harsh spotlight that was cast on the clinical trial world.

In a 2002 article in Epidemiology Review, Jeremy Sugarman, MD, MPH, a professor of bioethics and medicine at the Johns Hopkins School of Medicine, wrote, "It is critical to ensure that research is conducted responsibly throughout the entire study cycle, from the way participants are selected to the way data are entered, analyzed, and reported. Attention to each aspect of research conduct is necessary to the success of the scientific enterprise and to the protection of study participants and others from unnecessary harm."

Conundrum: At what point does cost sharing backfire? A 20 percent coinsurance on a $20,000 therapy may work actuarially but could foster patient nonadherence.

The issue, of course, is complicated, because patients - especially suffering patients - are willing to try something new, even when physicians acknowledge that a complete side-effect profile isn't known, according to Eric Wickstrom, PhD, a professor of biochemistry and molecular pharmacology at Jefferson Medical College. This headlong rush by ailing volunteers, he says, necessitates rigorous protection by review committees, even before patients see an informed consent form. This is especially true, he says, for some vaccines and gene therapy.

"Gene therapy hasn't really worked yet, and it necessitates a great deal of care," explains Wickstrom.

The Gelsinger episode, though, raised another sticking point - financial conflicts of interest. In this case, the lead researcher, James Wilson, MD, failed to disclose in the consent form that he had founded a company that stood to profit from the research.

Arthur L. Caplan, PhD,2 who heads the Center for Bioethics at the University of Pennsylvania, stressed in a recent interview with Biotechnology Healthcare that researchers must ensure that clinical trials are not distorted by incongruous arrangements. In addition, he says, volunteers should not be recruited in a manner that would suggest that they are being paid bribes, as opposed to reimbursement for legitimate expenses.

2. Affordability

The rising cost of healthcare - and the cost of medications in particular - is a political hot potato and will remain so. No matter what the U.S. Food and Drug Administration might say or attempt, a large swath of the public, their federal representatives, and their governors do not seem to believe the pharmaceutical industry's argument that research and development are funded by today's prices and that price controls could retard R&D.

The ethical concerns are likely to get still more heated when the value of expensive biotech treatments for chronic illnesses is debated. After all, a needed pill for cholesterol might cost $3 daily, which amounts to nearly $1,100 per year. Compare that to a biologic that carries a $20,000-per-year price tag - or something even more costly.

"Affordability is, arguably, an issue across the board," says Jeff Kimmell, RPh, vice president of healthcare services and chief pharmacy officer at drugstore.com, in Bellevue, Wash. "In the United States, we say we want the best [treatments]. But it's also an ethical dilemma. At what point will people say 'Enough is enough?'"

This may place payers and purchasers, who are already struggling with the question of how much cost sharing is appropriate, on the defensive. Insurers and employers juggle actuarial concerns with the risks of patient nonadherence and its potential for poor clinical outcomes when coverage decisions are made. The ethical questions do not fit neatly into this decision-making process but, rather, transcend it.

What happens when some patients can't afford the out-of-pocket share of a given treatment? What if an insurer declines to add a biologic to its formulary because of its acquisition cost? What happens when a patient on an expensive chronic therapy maxes out his lifetime insurance benefit? Such instances may not be the norm, but their possibility disturbs some experts who see a pivotal clash between patients and profits.

"It's certainly an economic issue if biologics are priced so high that some patients are priced out of the market," says Sean Nicholson, PhD, assistant professor of policy analysis and management at Cornell University. "Perhaps an insurer may not cover a particular therapy. If there's nothing else the patient could take to save his or her life, or to improve quality of life, that's a dilemma."

3. Privacy

Talk about a Pandora's box. Protecting patient privacy is a growing concern, thanks to technology that is making it possible to decode the human genome. But as scientists become adept at deciphering a person's genetic composition, it is increasingly likely that compromising information about a person's future health is going to become available.

This creates enormous problems. For instance, it may become possible to know that a 5-year-old is going to develop serious heart disease later in life, but does a prospective employer have the right to know that? How will this knowledge affect the individual's ability to obtain a job, insurance, or a mortgage? Should such information be available to insurers and others? This is a thorny problem destined to become only thornier.

"Take a genetic test that comes back positive, and the biologic treatment of the patient costs $1 million," says F. Randy Vogenberg, RPh, PhD, senior vice president and national practice council leader at Aon Consulting, in Providence, R.I. "What does an insurer do with that knowledge? It can't tell the employer, but it does have a fiduciary responsibility as the health plan to remain within stated benefit coverage premiums and costs.

"It's going to be very tricky for health plans to deal with this. And it won't just be on a case-by-case basis, but it will come up during plan renewals and bidding."

Sound far-fetched? Not really, says Vogenberg. "Generally speaking, everybody so far has stuck their heads in the sand. HIPAA offers some protection, but as diagnostics expand, this issue will bubble to the surface. And probably, litigation will determine some boundaries."

To nip the problem in the bud, some attempts are being made to put rules in place now. An American College of Physicians working group published one such effort in the Annals of Internal Medicine in July; an article titled "Ethics in Practice: Managed Care and the Changing Health Care Environment" spelled out a statement of ethical principles for health plans, purchasers, and physicians. One section opined that "All parties have an ethical obligation to protect the confidentiality of patient health care information. In general, identifiable patient information should not be shared without the patient's consent - except where the health and safety of individual persons or the public may be threatened, or as required by law."

Conundrum: What does an insurer do with the knowledge that a person is predisposed to a condition that costs $1 million to treat? It can't tell the employer, but it has a fiduciary responsibility to remain within stated benefit coverage premiums and costs.

Even this statement, however, leaves open the question about the extent to which an individual's rights may be eclipsed by societal needs. This is especially true in the wake of the Patriot Act. In reflecting public fears about terrorism, the federal law is causing a raging debate over an individual's right to privacy and the safety concerns of a society at large. With legal challenges pending, it is easy to see how even the ethics statement for managed care may cause difficulties.

4. Stem Cell Research

This one shouldn't be a surprise. Stem cell research is anathema to the religious right and worked its way into the recent presidential election.

To circumvent the funding roadblocks in Washington, D.C., some states - such as New Jersey and California - are looking past ethical objections and taking serious steps to foster establishment of stem cell research centers. In California, birthplace of the biotech revolution and a state hard hit by the dot-com collapse, voters sized up the potential economic value of stem cell research and overwhelmingly passed Proposition 71, which guarantees $3 billion in state funding over the next decade.

It is somewhat ironic that a scientific area of research based on trials and data could be reconfigured into an emotional issue. The debate, of course, pits people who believe the research may one day find cures for diseases against others who say it violates human life.

On one side, the American Society of Clinical Oncology issued this policy statement in 1999: "Whether privately or publicly funded, researchers should be mindful of the ethical issues that may be raised when research involves embryos, fetal tissue, cloning, or other controversial questions. Nevertheless, medical research often requires a balancing of perceived risks against potential benefits. The tremendous potential of stem cell research for the treatment of diseases ... means that the balance of benefits and risks is now clearly in favor of going forward with the research, even if it involves ethically sensitive areas."

On the other hand, the founding statement of Do No Harm - The Coalition of Americans for Research Ethics argues: "Stem cell research promises great good and is a worthy scientific priority as long as we pursue it ethically. Obtaining stem cells from people without seriously harming people in the process can be ethical. However, obtaining stem cells from human embryos cannot be ethical because it necessarily involves destroying those embryos."

For now, say experts, the debate is largely hypothetical for insurers.

"Although it is still early in the process, it is likely that the managed care industry will continue to monitor this ethically sensitive area, particularly as the potential benefits and costs begin to outweigh the risks," says Mitchell P. DeKoven, manager of reimbursement services at PharmAnalysis Group, MED-TAP's Center for Pricing & Reimbursement, in Arlington, Va.

5. Defending the United States Against Bioterrorism

Security is hugely important, and public fears over terrorism are unlikely to diminish. In response, the federal government wants Project Bioshield to spur the development of treatments, including preventive medications and vaccines, that would be available in sufficient quantities to protect the largest possible number of people.

The congressional impetus is obvious, according to Chuck Ludlam, general counsel on the staff of Democratic Sen. Joseph Lieberman of Connecticut. In a recent speech, Ludlam said that economic losses caused by a successful bioterror attack in a densely populated area could be massive.

But Project Bioshield also raises ethical problems, experts say. For starters, the investment will be large and probably will grow over time. At a time when deficits are growing, generous funding for bioterrorism research suggests that funding for other public health priorities - such as diseases that are becoming more prevalent - may suffer.

"If we fund this, what are we not funding? And how do you divide the pie?" asks David Krause, MD, senior vice president of clinical research and medical affairs at Vicuron Pharmaceuticals, in King of Prussia, Pa. "And that's just part of the problem. We don't know if anybody will ever die from bioterrorism. We don't know what the threat is. Can we ever predict all the possible terror threats and provide protection against them?"

Krause isn't alone in observing that there is an unpredictable number of portals for bioterrorists to wreak havoc on the United States. The Infectious Diseases Society of America wants the scope of Project Bioshield extended to include all areas of infectious disease research and development, especially for antibiotics to treat existing drug-resistant infections.

"There is an inextricably linked, synergistic relationship between research and development efforts needed to protect against both naturally occurring infections and bioterrorism agents," said John Bartlett, chief of the division of infectious diseases at the Johns Hopkins School of Medicine and chair of an IDSA task force, in testimony given recently before the U.S. Senate Committee on Health, Education, Labor and Pensions, and the Senate Judiciary Committee.

On the positive side, bioterrorism research may yield the same sort of consumer-product payoff NASA research did - in this case, Ludlam noted, possibly indirectly helping to find medicines to treat infections with HIV and viruses that cause hepatitis, Lyme disease, polio, West Nile, and the common cold. The effort also could lead to medicines for bacteria, such as those causing tuberculosis, pneumonia, salmonellosis, malaria, sepsis, or sexually transmitted diseases.

A subsidiary ethical issue to bioterrorism research, says Krause, is clinical testing. Human subjects couldn't be used in smallpox trials, for instance, so surrogate markers are required. This can make it difficult to determine the effectiveness of an antiterrorism agent.

"We're talking about an expensive insurance policy and, like any insurance policy, you hope you never have to use it," says Krause. "You want it in your plan, but at the same time, it's not a slam dunk."

Footnotes

¹ About our methodology: The editors selected and ranked the five broad topics in this article on the basis of the results of a questionnaire distributed to the Biotechnology Healthcare editorial board. The editorial board (see page 3) represents numerous disciplines, and includes physicians, pharmacists, researchers, ethicists, insurers, managed care executives, employers, government policy experts, and consultants.

² Astute readers will recall that Caplan had a courtesy appointment to Wilson's department and provided him with informal advice about enrolling patients in gene therapy trials. Caplan, who has been active in his career in pushing informed-consent reforms, ultimately was found to have no responsibility in the Gelsinger case.

Articles from Biotechnology Healthcare are provided here courtesy of MediMedia, USA

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The 5 Most Pressing Ethical Issues in Biotech Medicine

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Biotech healthcare is going through what every other emerging scientific discipline experiences - the challenge of defining its ethical boundaries. Research, costs, and privacy issues spawn concerns that third-party payers, employers, providers, and policy makers will face for years to come.

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The best polo horses can be cloned to create a team of 7 or nine. These horses are identical and are cloned off of the best polo horse the owner has. This takes away from the spirit of competition and should not be allowed.

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You don't expect to hear that some of the most cutting-edge biotechnology is now part of the elite game of polo, "the ancient sport of kings."

But on a trip to Argentina last December, we went to a big polo match -- and discovered that several of the champion horses on the field were clones.

It's a big day in Buenos Aires - the final match in this year's World Cup/Super Bowl of polo called the Argentine Open - with the usual pageantry, the tango included. In polo, the horses, called ponies, are just as important as their riders.

The two teams are La Dolfina in white and Ellerstina in black.

Each team has four players who ride as many as a dozen horses during matches. All of the players today have reached the highest ranking in the sport - a 10 goal handicap.

The player generating the most interest is the man in the blue and white helmet, Adolfo Cambiaso. He's led his team to victory for the last five years. At 42, he's the Tom Brady of polo.

Adolfo Cambiaso: I love the sport that I do. I love polo. I love horses. And so I try to be the best.

Lesley Stahl: You are number one in the world in your sport. That's stunning, isn't it?

Adolfo Cambiaso: It's strange. When they say it to you, you don't feel like it but...

Lesley Stahl: How long have you had this title? How many years?

Adolfo Cambiaso: For 22 years.

Lesley Stahl: You've been the best in the world for 22 years?

Adolfo Cambiaso: That's what they say.

At age 25, Adolfo decided to create his own polo team called La Dolfina, and a breeding business from scratch. Today he has nearly a 1000 horses that are fed a special diet of plants and grasses grown on his massive farms.

Adolfo Cambiaso: If they have a little pain somewhere I dig a swimming pool for them just to swim.

A swimming pool for the horses, where they do laps and stretch out their sore muscles.

Lesley Stahl: And they like it?

Adolfo Cambiaso: They love it.

Lesley Stahl: They do? They like to--

Adolfo Cambiaso: They love it. They jump on. It's amazing.

His most prized horse for a long time was named Aiken Cura. But at the Argentine Open 12 years ago, Aiken Cura's leg was broken. And Adolfo was devastated.

Adolfo Cambiaso: More than anything, I say, "Save this horse."

Lesley Stahl: He was your favorite--

Adolfo Cambiaso: Yes.

But the horse could not be saved. Before they put him down, Adolfo made a fateful decision: he asked his veterinarian to save some of the horse's skin cells. He thought that one day he could bring Aiken Cura back to life through cloning.

Adolfo Cambiaso: I was really sad and I say cloning should work --

Lesley Stahl: How did that come into your head?

Adolfo Cambiaso: I don't know. I decided to keep some cells from him, just in case years later-- cloning-- is normal.

He remembered Dolly the sheep, the first cloned mammal. Since then, scientists have cloned cows, pigs, goats, and in 2003, the first horse.

Biologist Adrian Mutto, one of the first scientists to clone in Argentina, showed us the process: he starts with an egg extracted minutes earlier from a mare.

Dr. Adrian Mutto: You can see here, this is an egg. And with that needle, we eliminate all DNA of each egg.

Next, he replaces it with the DNA of the horse they want to clone.

Dr. Adrian Mutto: The next step is introduce again into the, into the egg the needle. This is the DNA into the egg.

Lesley Stahl: You did it?

Dr. Adrian Mutto: Yeah This is our cell and this is the egg.

Lesley Stahl: And that's it.

Dr. Adrian Mutto: That's it.

The new embryos are then incubated for one week. No sperm has been involved.

Dr. Adrian Mutto: We don't need the sperm.

Lesley Stahl: There's no male--

Dr. Adrian Mutto: Yes. Yes, no male here. Only me.

Lesley Stahl: But-- but that's incredible. The-- there-- it's-- so it's not a male-female reproduction at all.

Dr. Adrian Mutto: Yes.

Lesley Stahl: You're just taking a cell from whichever. Could be a mare or could be a male horse--

Dr. Adrian Mutto: Yes.

Lesley Stahl: And you're putting it in this egg--

Dr. Adrian Mutto: The cell, into the egg.

Lesley Stahl: No sex at all?

Dr. Adrian Mutto: And-- we, no. Poor horses.

The incubated egg is implanted in a surrogate mare who gives birth to the clone -- like this one that's 3 weeks old.

Cloning represented a business opportunity to this man, Texas oil heir and polo enthusiast Alan Meeker. He had long dreamt of building a fleet of champion horses, and now had a way to do it.

Alan Meeker: I did some short math and I realized it would take 50 years and about $100 million to do what I wanted to do. And I thought, "Well, why don't I just clone a bunch of horses, really, really good horses.

In 2009, Meeker founded a horse-cloning business and, a year later, licensed the technology that was used to clone Dolly the sheep.

Alan Meeker: "Okay, now-- now I need to find the best horses." So I put together an idea of licensing the genetics from the very best-- breeders in polo. I knew some people that knew Adolpho.

Lesley Stahl: Was he considered one of the best breeders? Not just the best players, but also one of the best breeders?

Alan Meeker: Breeders and owners of horses. His horses were performing better than anyone else.

Lesley Stahl: When Alan first approached you about cloning?

Adolfo Cambiaso: And I say yes the first day.

Lesley Stahl: Immediately.

Adolfo Cambiaso: Yes.

Lesley Stahl: And, "Guess what," you said, "Alan, guess what I have"?

Adolfo Cambiaso: Yeah. I wanna tell him that I have-- cells from a horse that I really loved that I would love to clone.

It took a while to get it right - one attempt failed, but after two years, Adolfo got his wish. The birth of a clone of his beloved Aiken Cura who grew into this magnificent, healthy horse, almost exactly like the original in strength, athletic ability and temperament.

Adolfo Cambiaso: When I saw him, I couldn't believe it.

Lesley Stahl: Did you know by just looking-- and of course it was a little foal at that point--

Adolfo Cambiaso: Yes. It was. But...

Lesley Stahl: You could tell?

Adolfo Cambiaso: To make sure, I took some hair from him, and I bring him back to Argentina to do the DNA.

Lesley Stahl: To double-check.

Adolfo Cambiaso: To double-check it was him.

At the same time, he decided to clone another horse -- his biggest star -- a mare called Cuartetera.

Now 17 years old, the original has been playing polo since she was 4 - a year younger than most polo ponies - simply because she took to the game so quickly.

Adolfo Cambiaso: I think she's born to play, you know? There is those horses in life or like soccer players like Messi. It's not many.

Lesley Stahl: Like you.

Adolfo Cambiaso: No. I don't know. No. But what I'm saying, this horse is amazing.

He took us to the barn where Cuartetera lives with eight of her clones.

Adolfo Cambiaso: You see those-- these two little points--

Lesley Stahl: Yeah.

Adolfo Cambiaso: From this little point is where you make all these horses.

Lesley Stahl: This is where they took the cells-

Adolfo Cambiaso: Exactly.

Lesley Stahl: --to make the other--

Adolfo Cambiaso: To make the other. Because of her you get all these ones.

Lesley Stahl: And that was what you were thinking?

Adolfo Cambiaso: Yes.

Lesley Stahl: "I'm gonna"--

Adolfo Cambiaso: That was my dream but everybody was--

Lesley Stahl: --"clone the best."

Adolfo Cambiaso: --saying that I was crazy. And I like it right now because I'm having a good time hearing those people.

Lesley Stahl: Yeah, they're saying, "He's not so crazy anymore"-

And look what he's done: in seven years, he and his partners have created more than a dozen clones of Cuartetera.

Dr. Adrian Mutto: It's incredible for me. I never lose my, wow, this is-- my production. This is my equine daughter.

Dr. Mutto, who was hired as the lead scientist in Adolfo's cloning business, took us to see the Cuartetera clones he thinks of as his children.

Dr. Adrian Mutto: This is-- Cuartetera number five.

Lesley Stahl: Oh my God.

Dr. Adrian Mutto: This is number four. Number three. Number nine.

Lesley Stahl: Oh my God.

Dr. Adrian Mutto: Number six.

Lesley Stahl: You can tell which one.

Dr. Adrian Mutto: Yeah, be--

Lesley Stahl: You're not reading anything--

Dr. Adrian Mutto: --because I know her by the-- but-- they are-- are all clones.

Dr. Adrian Mutto: Yes. Right now, we have 14.

Lesley Stahl: Just from Cuartetera?

Dr. Adrian Mutto: 14, and next year, 10 more. And 2019, 10 more.

In all, there are more than 100 clones from several of their best horses. In each case, he said the clones are strikingly similar to the originals in disposition, athletic ability and appearance. But not exactly. For example, the Cuartetera clones all have white markings, but with different shapes and in different places, some on the face; some on the ankle.

But all the Cuarteteras seem to have inherited the original's calm, self-contained personality.

Lesley Stahl: So the genetics include this temperament?

Adolfo Cambiaso: Yes.

Lesley Stahl: And do the clones get along with each other?

Adolfo Cambiaso: Yeah, because they live together all year long. So-- from here, they go to the farm together, then they move in blocks. If you take one out of them, they are looking for it.

Lesley Stahl: They miss the one that you take out.

Adolfo Cambiaso: Yeah.

Lesley Stahl: Did the clones have any special health issues?

Adolfo Cambiaso: No.

We talked to scientists at the National Institute of Health and were told there is no evidence that cloned animals suffer disproportionate health problems, though they have a slightly higher infant mortality rate. At first, many of Adolfo's cloned embryos died during gestation. But they refined their technique and now tell us they have an 85 percent successful birth rate and have not experienced any health problems.

Lesley Stahl: So as far as you are thinking, they're exactly the same in health, longevity.

Adolfo Cambiaso: Si.

Lesley Stahl: Ability to play the game, all of it.

Adolfo Cambiaso: Similar. Similar. Not exactly the same...

Lesley Stahl: What are the differences?

Adolfo Cambiaso: There is some that are a little bigger. Some eat more, some eat less. Or they move a little bit different. But the mind are really similar. The good thing about it, they are machines, all of them.

Machines -- that's polo talk for horses that never quit.

But how would they perform in competition? At the final at the Argentine Open, Adolfo gambled that his Cuartetera clones would be as good as the original and, for the first time, he rode them almost exclusively.

Regulators of thoroughbred horse racing worldwide have taken a firm stand against cloning. But there is no such prohibition in polo and so cloning is spreading to teams beyond Adolfo Cambiaso's. It raises some thorny questions: does cloning give a team an unfair advantage? Is it ethical? And where will it lead?

In December, at the final match of the Argentine Open in Buenos Aires, one team rode clones while the other refused to. The competition was as much about the merits of cloning as it was a sporting contest.

Out of more than 850 professional polo players, Argentine native Adolfo Cambiaso - wearing the blue and white helmet and the jersey marked number one, is the best player in the world. He's held that ranking for 22 years and is now leading a cloning revolution. He's cloning his best horses; the one he's riding is a clone. He's competing on them - and winning.

Lesley Stahl: When you're on one of the clones playing, is there a special feeling? Knowing that h-- you know, this is something that was your idea, you brought it to life--

Adolfo Cambiaso: Yeah. In this stage of my career-- the last couple of years for me to play and prove that the clone works and play with Cuarteteras and everything is an extra motivation for myself, for sure.

Lesley Stahl: I don't know that you need extra motivation.

Adolfo Cambiaso: Yeah, I do. I do.

He's created 14 clones of Cuartetera, his very best horse - a 17-year-old mare who is fast, easy to direct and can turn on a dime. She was honored last year as the best polo horse in history. Her clones seem to be just as gifted.

Lesley Stahl: Are they all as good - I want to call her Mama. I don't-- That's probably not the right word.

Adolfo Cambiaso: The original. Yeah.

Lesley Stahl: Are they as good?

Adolfo Cambiaso: I already won two Argentine Opens with the clones. So they will end up being as good as her I think.

Cuartetera's clones are identified by numbers.

Lesley Stahl: Shouldn't they have names?

Adolfo Cambiaso: They have names.

Lesley Stahl: Well, they don't.

Adolfo Cambiaso: Cuartetera.

Lesley Stahl: Yeah, but-- m--

Adolfo Cambiaso: Cuartetera one, two, three, four--

Lesley Stahl: Was that your idea?

Adolfo Cambiaso: Yes. Because I-- I believe that she is a Cuartetera. All of the ones that I ride, they are Cuarteteras.

Lesley Stahl: So you actually think--

Adolfo Cambiaso: She's Cuartetera when I play.

Lesley Stahl: When you're-- so six is Cuartetera, is--

Adolfo Cambiaso: Is Cuartetera.

Lesley Stahl: And when you're on nine, it's the same thing?

Adolfo Cambiaso: Cuartetera.

Lesley Stahl: But she's an individual.

Adolfo Cambiaso: Yes. But the DN-- DNA, it's a Cuartetera.

Lesley Stahl: But when you have identical twins, they each get a name.

Adolfo Cambiaso: But this is not twins, it's a clone.

They can now create 100 clones a year and they're using them in Adolfo's already successful breeding business. They mate the clones with champion horses and sell their foals for up to $250,000. But they never sell the clones.

Adolfo Cambiaso: You sell the clone, you sell the blood, you sell the line, you sell the DNA, you sell everything if you sell a clone.

Ernesto Gutierrez: We keep the key of the genetics and this was, I think, the good business to make that decision in the past.

The idea of never selling the clones came from Ernesto Gutierrez, a shrewd Argentine businessman, who became a third partner in the cloning venture. The cloning operation was set up here on his 500-acre property outside Buenos Aires that includes three polo fields, and a nursery where the clones are born. They are carried by surrogate mares who treat them like their own.

Lesley Stahl: Are these all cloned babies in here?

Ernesto Gutierrez: All cloned babies? Yeah.

Lesley Stahl: And these are the surrogate mothers? And does the mother think it's totally her baby?

Ernesto Gutierrez: Totally, totally. Look at that.

Gutierrez took us back to see the newest one - that 3-week-old clone of Cuartetera who has her own nurse.

Lesley Stahl: Oh, look how sweet. And frisky. Oh, look at that, oh my.

Not everyone in polo thinks cloning is a good idea - including Adolfo Cambiaso's main rival and opponent at the final of the Argentine Open.

Facundo Pieres: There's a lot of guys cloning. But I think that they have to be careful, you know because, the thing is that they're opening too much, you know? I mean--

Lesley Stahl: Pandora's Box. You know what that means? They let-- opened the lid. And all the problems come out-

Facundo Pieres: Exactly.

Facundo Pieres is number two in the world, right behind Adolfo. He showed us what he can do, like dribble a three-inch ball in the air while galloping down the field 20 miles an hour. He's the captain of Ellerstina, an old-school team made up of three brothers and a cousin. They are committed to keeping it a family enterprise.

Lesley Stahl: Do you ever get angry at each other?

Facundo Pieres: Yes. Yes, but in a good way. Never-- never bad.

His team is headquartered at another sprawling estate where they operate a multi-million dollar breeding business selling foals and embryos. They believe they can produce better horses through their breeding practices - by mixing the DNA of two different horses, rather than by replicating just one.

Facundo Pieres: We wanna keep it this way. What we have here is amazing

Lesley Stahl: In polo, what's more important, the horse or the player? I was told that it's 80 percent the horse. Sorry--

Facundo Pieres: Yes, I-- I agree. No--

Lesley Stahl: No offense.

Facundo Pieres: No, I agree. I agree. I totally agree. I think that the horse is. But of course, you need to have a little bit of-- of talent and ability and-- and experience in the head, you know?

Facundo's team - Ellerstina - has made it to the finals the last 4 years, but lost each time to Adolfo Cambiaso's team. Fueling the rivalry on the field is a bitter history between them: Adolfo played on the Ellerstina team for 9 years.

Adolfo Cambiaso: Because of what happened, that I left Ellerstina and the rivalry is there--

Lesley Stahl: Intense.

Adolfo Cambiaso: Yeah.

Lesley Stahl: To this minute.

Adolfo Cambiaso: Yeah.

Lesley Stahl: Do you feel it too?

Adolfo Cambiaso: But it's fun.

Lesley Stahl: Oh, you like it--

Adolfo Cambiaso: You gotta have rivalry to be better player too.

There's more. Before he left Ellerstina, Adolfo bought Cuartetera, as an embryo, from the Pieres family... the very horse he is now cloning to compete against them.

Adolfo Cambiaso: I was lucky to end up with Cuartetera.

Lesley Stahl: You cloned from the best horse in the world.

Adolfo Cambiaso: But she's born on my farm. I create her.

Lesley Stahl: There are people who object to cloning on religious grounds... Or on moral grounds. So what is the answer when people challenge you? When they say, 'Man should not be doing this' because of these difficult spiritual questions?

Adolfo Cambiaso: I don't see it, I don't see it wrong, to be honest. I'm just-- doing something for-- to improve my game, my sport. And I think the Cuateteras did improve my game, my sport. And I'm not going farther than that.

Lesley Stahl: But, is there an unfair advantage in terms of the game, in terms of the sport?

Adolfo Cambiaso: No, because everybody's able to clone. Now everybody's kind of trying to start cloning. So the advantage is that I did it seven years ago.

Lesley Stahl: So in 30 years, people will still be riding Cuartetera?

Adolfo Cambiaso: Yes.

Lesley Stahl: And so it could go on forever.

Adolfo Cambiaso: Yes. Yes.

Alan Meeker, the Texas businessman who is Adolfo's cloning partner, is well aware of the controversy around cloning technology in the U.S. and the ban against it in thoroughbred horse racing.

Lesley Stahl: Is a really good polo player-- does he have-- an unfair advantage if he's on a clone of one of the best-- polo horses ever?

Alan Meeker: Of course. Horses are 80 percent of the game anyway. So if Facundo Pieres finds a horse that is better than Cuartetera than he has an advantage over his competitor.

Lesley Stahl: But he'll have only one. And that horse will get tired, and he'll have to switch to another horse in the game, whereas Adolfo will have 8.

Alan Meeker: Right.

Lesley Stahl: So it's still an advantage.

Alan Meeker: Right.

Lesley Stahl: Is that fair?

Alan Meeker: Under the rules it's fair. There's no restriction on...

Lesley Stahl: I know but the game - but sportsmanship, just the nature of the game. Has this changed the very essence of the game of polo?

Alan Meeker: No. I think what it's done is probably raise the bar.

Lesley Stahl: You're going to have to clone.

Alan Meeker: Could be. Yes.

Lesley Stahl: Do you have any moral problems with cloning a human being?

Alan Meeker: Yes. I disagree with it. I know a good reason, lots of good reasons to clone-- body parts, like hearts and lungs and pancreases, if it could be done in a productive manner, that can save lives. But I've been asked by some of the wealthiest people on planet earth to clone a human being and we--

Lesley Stahl: You have?

Alan Meeker: Absolutely.

Alan Meeker: And the answer is always-- a resounding "no."

Lesley Stahl: Well, they must have a reason.

Alan Meeker: And they won't give it to me.

Lesley Stahl: They don't tell you why?

Alan Meeker: No.

Lesley Stahl: I'm thinking if science can do it, science will do it and maybe one day, you know, they'll be clones and we'll laugh at all the people who were questioning the morality of it now.

Alan Meeker: Someday someone will do it--

Lesley Stahl: Yes.

Alan Meeker: And we will either laugh or we will cry. But I'm not gonna be the one to take that-- that leap.

Lesley Stahl: It could be done today.

Alan Meeker: Yes.

Lesley Stahl: I assumed there'd be a big difference between a horse and a human. Lots of differences.

Alan Meeker: Surprisingly little. Yeah. Surprisingly little.

At the final match at the Argentine open, Adolfo's team and the clones were expected to win, but seven minutes in, Facundo's team was ahead, three goals to one.

Adolfo's team fought back; at halftime, the score was, the cloners, 7; the breeders, 6. It was so tense that at times it was as quiet as a tennis match.

The end of the game was thrilling: as expected, Adolfo's team was ahead, 13 to 10, but then Facundo's team in a final blast came back to tie the match.

Adolfo Cambiaso: I never think I'm-- I gonna lose. I never.

Lesley Stahl: Well, we saw you right before the overtime.

Adolfo Cambiaso: Yeah.

Lesley Stahl: And here you are. Like that.

Adolfo Cambiaso: In that moment, I was trying to think, "Which is the best horse for that moment?"

He debated, should it be Cuartetera 9? Or 5? Finally he picked number 6.

In the first minute of the sudden death overtime, Facundo's team lost control of the ball.

Adolfo's team recovered and Adolfo on his mighty Cuartetera 6 outran everyone and whacked the ball setting up the winning shot.

Watching, you had to wonder: was it the clones or the world's best player that made the difference?
Produced by Sarah Koch, Nieves Zuberbühler and Terry Manning. Associate producer, Chrissy Jones.

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The horses of an Argentine polo club may look normal, but the way they came into existence is anything but conventional

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CRISPR Has worked to edit heads off diseases and started testing this on mice. Mice actually did better than with a drug on reversing a disease.

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Two studies published yesterday (October 9) in Nature Medicine report success using modified CRISPR-Cas9 gene editing to prevent or cure two inherited diseases in mice. In one study, researchers corrected the gene mutation that causes phenylketonuria (PKU) in the livers of adult mice; in the other, editing short-circuited a condition similar to the human disease HT1 in the livers of fetal mice.

In the PKU study, carried out at ETH Zurich in Switzerland, researchers bundled a CRISPR-Cas9 system plus the enzyme cytidine deaminase into an adeno-associated viral vector and injected it into the livers of mice with defective genes for phenylalanine hydroxylase. (A lack of functional phenylalanine hydroxylase causes a toxic buildup of phenylalanine in patients with PKU unless they're diagnosed soon after birth and put on a special diet.) The gene-editing homed in on and changed a single base pair in the genomes of the mouse liver cells, reversing symptoms of the disease in the animals.

In the HT1 study, researchers at the Children's Hospital of Philadelphia and the Perelman School of Medicine at the University of Pennsylvania paired CRISPR-Cas9 with base editor 3, which converts a single DNA base into another DNA base. Rather than targeting the disease-causing gene mutation, the team used the editing tool to disable a different gene, HPD. This prevented toxic products of the breakdown of tyrosine from building up in the body, as they normally would in people with HT1 or mouse models of the disease. The gene-editing machinery was packaged into an adenovirus vector and injected into a vein that feeds the liver in fetal mice. After birth, the gene-edited mice were compared with untreated controls and with mice that received a drug normally used for the disease.

"We weren't expecting it, but the genome-edited mice did much better" than mice treated with the drug, coauthor Kiran Musunuru of the Perelman School of Medicine tells . "They survived longer and gained more weight."

Robin Lovell-Badge, a stem cell biologist at the Francis Crick Institute who was not involved in either study, tells The Guardian that the HT1 work comprises "important proof-of-principle experiments that suggest that genetically inherited liver diseases might be avoided by correcting a defect before the effects of the mutation will have compromised liver function in the newborn child."

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CRISPR Editing Heads Off Disease in Mouse Livers

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Separate proof-of-concept studies report success against two inherited diseases.

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Surgeons have created a fetus with cells from the mother's bone marrow. The child was carried to term and delivered with almost no birth defects.

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NOAH BERGER/UCSF

Pediatric surgeons at the University of California, San Francisco, have treated a second-trimester fetus with stem cells taken from her mother's bone marrow. The baby, born in February, was the first patient enrolled in the world's first clinical trial using stem cells transplanted prior to birth. She is "apparently healthy," despite living with a deadly genetic disease called alpha thalassemia, according to a statement from the university.

Tippi MacKenzie, a pediatric surgeon at the University of California, San Francisco (UCSF) Benioff Children's Hospital, led the team that performed the transplant. "It is too early to say how effective the stem cell transplantation will be, but we are encouraged by how well she and her mother have tolerated the treatment," she says in the statement. "Her healthy birth suggests that fetal therapy is a viable option to offer to families with this diagnosis."

The baby, Elianna Constantino, was born on February 1 to Nichelle Obar after five blood infusions and one stem cell transplant over the course of four months in a clinical trial funded by the California Institute for Regenerative Medicine. The trial began in 2016 to investigate using stem cells to treat thalassemia. Constantino has a lethal form of it, called alpha thalassemia major, which is caused by abnormalities on the HBA1 and HBA2 genes, reduces the body's ability to produce hemoglobin, according to the National Institutes of Health's Genetics Home Reference, and can cause swelling of the liver or heart. Babies with alpha thalassemia typically die before birth or are stillborn.

During pregnancy, a routine ultrasound performed at a medical center in Hawaii, where the family resides, detected the fetus had an enlarged heart. According to the UCSF statement, intrauterine blood transfusions were required to treat the swelling before the stem cell transplant could be performed. The transplant was given during one of the transfusions.

"Elianna's doing great," Obar tells The New York Times. "I'm not disappointed at all. If it works, great. If it didn't, we're O.K. with it. We'll celebrate all the little accomplishments. I'm glad we did it."

"Once universally fatal, thalassemia can now be managed as a chronic disease," says Elliott Vichinsky, a hematologist and the founder of the Northern California Comprehensive Thalassemia Center at UCSF Benioff who is overseeing the baby's treatment, in the statement. "In utero stem-cell transplantation may take it one step further: as a disease that can be successfully treated before birth."

"Everybody has a perfect donor when they're a fetus, and that's the mom," MacKenzie tells The New York Times. According to the article, she plans to perform a few more transplants, monitor the patients' progress, and then decide how to proceed.

Correction (May 31): The original version of this story erroneously stated the fetus was the first to receive a stem cell transplant in utero; she is the first to have recieved such a transplant as part of a clinical trial. The article has been corrected to reflect this; The Scientist regrets the error.

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In Utero Transplant in First Clinical Trial Successful

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Doctors treated the fetus, who has alpha thalassemia major, with cells from her mother’s bone marrow.

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This article talks about how biotechnology isn't something new and its been around and how its not about just breeding animals and plants its been around for thousands of years and be used for countless different things

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Biotechnology

Biotechnology

Biotechnology isn't something new - selective breeding to create more useful varieties of animals and plants is a form of biotechnology that human beings have used for thousands of years.

Biotechnology includes any use of science or technology to alter the characteristics of a particular breed or animal.

Biotechnology can be good or bad for animals - and it may also produce an answer to the ethical problems of experimenting on animals.

Transgenic animals raise a particularly difficult problem.

Human problems

Newspaper articles about the ethical problems of genetically engineered animals are usually concerned about the danger these animals may pose to human beings (usually to human health), rather than any implications for the animals themselves.

Animal rights

Genetic engineering and selective breeding appear to violate animal rights, because they involve manipulating animals for human ends as if the animals were nothing more than human property, rather than treating the animals as being of value in themselves.

Recent action to allow animals to be patented reinforces the idea of animals as human property, rather than beings in their own right.

Animal welfare

Biotechnology can be good for animals. Selective breeding and genetic engineering can benefit animals in many ways:

Improving resistance to disease

Breeding to remove characteristics that cause injury
eg selecting cattle without horns

But biotechnology can also be bad for animals - the good effects for the breeder can offset by painful side-effects for the animals:

Modern pigs have been bred to grow extra fast - some breeds now grow too fast for their hearts, causing discomfort when animals are too active

Broiler chickens are bred to grow fast - some now grow too fast for their legs

Regulating genetic engineering

Profitability is one of the major drivers of both selective breeding and genetic engineering.

If animal welfare is not to be compromised, research must be restricted by a counter-balancing ethical principle that prevents altering animals in a way that was bad for the animal.

One writer, Bernard Rollin, suggests that a suitable rule to regulate genetic engineering would be this:

This principle can easily be adapted to cover selective breeding.

Biotechnology and experimental animals

It's been suggested that genetic engineering may solve all the ethical problems of laboratory experiments on animals. The goal is to create a genetically engineered mammal that lacks sentience, but is otherwise identical to normal experimental animals.

Such an animal could not suffer whatever was done to it, so there should be no ethical difficulty in performing experiments on it.

Ethical problems:

This argument seems convincing, but do you feel comfortable about it?

Is there any ethical objection to creating genetically engineered human beings without sentience, and experimenting on them?

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Biotechnology includes any use of science or technology to alter the characteristics of a particular breed or animal.

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Basically, this article is stating that genetic modification is not as easy as we think, and it would be nearly impossible to genetically modify someones height, for example. "Some scientists estimate height is influenced by as many as 93,000 genetic variations. A recent study identified 697of them."

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This article toes over and talks about the ability to change a person genetics to benefit them in certain areas of life. For example better looks or becoming a superstar athlete.

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Much of what we currently know about the ramifications of genetic self-knowledge comes from testing for diseases. Once disease genes were identified, it became much easier to make a molecular or cytogenetic diagnosis for many genetic conditions. Diagnostic testing supplies the technical ability to test presymptomatic, at-risk individuals and/or carriers to determine whether they will develop a specific condition. This sort of testing is a particularly attractive choice for individuals who are at risk for diseases that have available preventative measures or treatments, as well as people who might carry genes that have significant reproductive recurrence risks. Indeed, thanks to advances in single-cell diagnostics and fertilization technology, embryos can now be created in vitro; then, only those embryos that are not affected by a specific genetic illness can be selected and implanted in a woman's uterus. This process is referred to as preimplantation genetic diagnosis.

For adult-onset conditions, ethical concerns have been raised regarding whether genetic testing should be performed if there is no cure for the disease in question. Many people wonder whether positive diagnosis of an impending untreatable disease will harm the at-risk individual by creating undue stress and anxiety. Interestingly, social science research has demonstrated that the answer to this question is both yes and no. It seems that if genetic testing shows that an individual is a carrier for a recessive disease, such as Tay-Sachs disease or sickle-cell anemia, this knowledge may have a negative impact on the individual's well-being, at least in the short term (Marteau et al., 1992; Woolridge & Murray, 1988). On the other hand, if predictive testing for an adult-onset genetic disorder such as Huntington's disease reveals that an at-risk individual will develop the disorder later in life, most patients report less preoccupation with the disease and a relief from the anxiety of the unknown (Taylor & Myers, 1997). For many people who choose to have predictive testing, gaining a locus of control by having a definitive answer is helpful. Some people are grateful for the opportunity to make life changes-for instance, traveling more, changing jobs, or retiring early-in anticipation of developing a debilitating condition later in their lives.

Of course, as genetic research advances, tests are continually being developed for traits and behaviors that are not related to disease. Most of these traits and behaviors are inherited as complex conditions, meaning that multiple genes and environmental, behavioral, or nutritional factors may contribute to the phenotype. Currently, available tests include those for eye color, handedness, addictive behavior, "nutritional" background, and athleticism. But does knowing whether one has the genetic background for these nondisease traits negatively affect one's self-concept or health perception? Studies are now beginning to address this question. For example, one group of scientists performed genetic testing for muscle traits on a group of volunteers enrolled in a resistance-training program (Gordon et al., 2005). These tests looked for single-nucleotide polymorphisms that would tell whether an individual had a genetic predisposition for muscle strength, size, and performance. The investigators found that if the individuals did not receive affirmative genetic information regarding muscle traits, they credited the positive effects of the exercise program to their own abilities. However, those study participants who did receive positive test results were more likely to view the beneficial changes as out of their control, attributing any such changes to their genetic makeup. Thus, a lack of genetic predisposition for muscle traits actually gave subjects a sense of empowerment.

The results of the aforementioned study may be surprising to many people, as one major concern associated with testing for nondisease traits is the fear that those people who do not possess the genes for a positive trait may develop a negative self-image and/or inferiority complex. Another matter bioethicists often consider is that people may discover that they carry some genes associated with physiological or behavioral traits that are frequently perceived as negative. Moreover, many critics fear that the prevalence of these traits in certain ethnic populations could lead to prejudice and other societal problems. Thus, rigorous social science research by individuals from diverse cultural backgrounds is crucial to understanding people's perceptions and establishing appropriate boundaries.

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genes and identity: human genetic engineering

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genetic engineering  eugenics  designer babies  genetic aristocracy

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Genes and Identity: Human Genetic Engineering

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Imagine having the option of custom making your body to possess the physical strength of Arnold Schwarzenegger or the endurance of Lance Armstrong. And what if you could choose to have your children look like Angelina Jolie or Brad Pitt, as well as have the intelligence of Albert Einstein?

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CRISPR’s Epic Patent Fight the article was basically about a fight that over who owns "one of the most common methods for editing the DNA in any living thing is finally drawing to a close. "

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CRISPR's Epic Patent Fight Changed the Course of Biology | Center for Genetics and Society

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The fight for Crispr-Cas9-which divided the research community and triggered an uncomfortable discussion about science for personal profit versus public good-has dramatically shaped how biology research turns into real-world products. But its long-term legacy is not what happened in the courtroom, but what took place in the labs: A wealth of innovation that is now threatening to make Cas9 obsolete.

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Research scientists around the world are now able to investigate the structural, cellular, and developmental intricacies of the human brain using bioengineered stem cell-based models called organoids. But the ethics to help guide researchers and regulators lag behind the technological capability to "grow" brains in a petri dish.

That is about to change as a group of bioethicists and scientists chart the unexplored territory of neuroethics, which is quickly emerging from the ability to bioengineer models of the brain. The study, called "The Brainstorm Project," will be led by Insoo Hyun, PhD, professor of bioethics at Case Western Reserve University School of Medicine. Hyun characterizes the project as a first step toward building a philosophical framework on which to base government policy and regulations.

"This is a new area of science for which we currently have few ideas on how to approach or map what works in terms of government and institutional regulation," Hyun says.

Launched this September in partnership with investigators from Massachusetts Institute of Technology (MIT), Harvard University and Stanford University, the two-year project is funded by a $569,410 grant from National Institutes of Health Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative.

"The organoid researchers in our Brainstorm Project are at the cutting edge of this science and this project is a novel opportunity to track their studies in terms of the emerging ethical issues," Hyun said. They are among the world's scientific leaders in this new field of neuroscience and are eager to explore ethical dimensions of their work alongside bioethicists."

The Brainstorm Project Working Group consists of bioethicists Hyun and his Case Western Reserve co-investigator, Aaron Goldenberg, PhD, MPH, associate professor and director of research for the Department of Bioethics, and co-director of the Case Western Reserve Center for Genetic Research Ethics and Law, along with Jeantine Lunshof, a bioethicist in the Church Lab at Harvard Medical School.

The bioethicists will interact weekly with scientists, including making laboratory visits to follow brain organoid experiments from the beginning, middle and end of studies at the collaborating universities.

"There are three major areas of organoid research," Hyun said. "In the first area, bioengineers try to maintain and grow the brain models for extended periods--months or years. They experiment with artificial circulatory and vascular systems over time until the organoids reach further maturity."

The second area of research consists of making more accurate brain organoids that have all the relevant cell types found in the natural human brain. The third area of investigation consists of performing signal readings to see how different brain regions function.

Multiple workshops will take place in year two of the project to explore findings in each of the three areas. The summary workshop will be held at Case Western Reserve, after which the team will make their report to NIH.

"An organoid is not a human subject, according to federal regulations," Hyun explained. "An organoid is an organ model in a petri dish. However, while people may view growing models of other organs, such as kidneys, in one way, they may view the idea of growing a model of a human brain more cautiously."

According to Hyun, close interaction between neuroscientists and bioethicists is something the BRAIN Initiative has wanted to accomplish. Hyun's approach--essentially co-developing a new field--is a focused effort to avoid the "silos," which are common in scientific fields.

Examining a range of ethics issues in this nascent field will help ensure public trust in science, as well, Hyun believes. The Brainstorm Project's ultimate goal is to develop greater awareness and understanding between scientists and ethicists and--beyond the duration of the project--to provide guidance for future management of ethical issues that may be unique to new areas of brain modeling research.

The Brainstorm Working Group partners from other universities are:

Jeantine Lunshof, PhD, a research scientist who studies conceptual and normative questions in the context of genome editing and an Ethics Consultant to the Church Lab at Harvard Medical School.

George Church, PhD, professor of genetics at Harvard Medical School and professor of health sciences and technology at Harvard and MIT, specializing in directed evolution of molecules, polymers, and whole genomes toward creating tools with applications in regenerative medicine and bio-production of chemicals.

Paola Arlotta, PhD, Harvard University Stem Cell Institute faculty member and co-director of the institute's Neuroscience Program, specializing in molecular laws that govern the birth, differentiation, and assembly of clinically relevant neuron types in the cerebral cortex.

Sergiu Pasca, MD, assistant professor of psychiatry and behavioral sciences in the Stanford Center for Sleep Sciences and Medicine and director of the Stanford Neurosciences Institute Stem Cells Core, specializing in groundbreaking studies on neural development.

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Study to explore bioethics of brain organoids

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Research scientists around the world are now able to investigate the structural, cellular, and developmental intricacies of the human brain using bioengineered stem cell-based models called organoids.

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“bioterrorism” refers to biological agents (microbes or toxins) used as weapons to further personal or political agendas. The article talks about the advancements and readily available biotechnology out there is controversial because the risk of using it for something like bioterrorism.

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What's the Problem?

The word "bioterrorism" refers to biological agents (microbes or toxins) used as weapons to further personal or political agendas. Acts of bioterrorism range from a single exposure directed at an individual by another individual to government-sponsored biological warfare resulting in mass casualties. Bioterrorism differs from other methods of terrorism in that the materials needed to make an effective biological agent are readily available, require little specialized knowledge and are inexpensive to produce. Until the aftermath of 9/11, few instances of bioterrorism were documented in the U.S.

A bioterrorist attack could be caused by virtually any pathogenic microorganism. The agents of greatest concern are anthrax (a bacterium) and smallpox (a virus). Both can be lethal. Anthrax is not communicable while smallpox is readily transmitted from person to person. In humans, the three forms of anthrax are inhalational, cutaneous and intestinal. Symptoms vary depending upon how the person was exposed but generally occur within 7 days of the exposure. Initial symptoms of inhalational anthrax may resemble the flu. If untreated, symptoms will progress to breathing difficulties and eventual shock. The incubation period for smallpox is 7 to 17 days following exposure. Symptoms include high fever, fatigue, and head and back pain. A characteristic rash follows in 2 to 3 days.

Who's at Risk?

In the U.S., the risk of contracting anthrax is extremely low. The intentional release of anthrax following the events of 9/11 resulted in only twenty-two recognized cases of cutaneous and inhalational anthrax. Any risk for inhalational anthrax due to cross-contaminated mail is also very low, even for postal workers. The possibility does exist, however, that if anthrax was dispersed in a public place, a large number of people could be affected. Smallpox has not occurred in the U.S. since 1949. If the virus was intentionally released, the number of people affected could run to the tens of thousands.

Can It Be Prevented?

Bioterrorism differs from other methods of terrorism in that the effects are not always immediately apparent. An attack may be difficult to distinguish from a naturally occurring infectious disease outbreak. The first evidence of an attack will be in hospital emergency rooms where the proper diagnosis will be essential in treating and preventing the spread of the disease. In the event of intentional anthrax distribution, people at risk should take a 60-day course of prophylactic antibiotics, either doxycycline or ciprofloxacin. Vaccination against smallpox is not recommended to prevent the disease in the general public. In people exposed to smallpox, however, the vaccine can lessen the severity of, or even prevent, illness if given within 4 days of exposure. The U.S. has a supply of vaccine for emergency use.

The Bottom Line

A story about bioterrorism carries inherent drama but also certain responsibilities. A story can raise concerns and heighten the public's awareness of the topic or it could cause alarm and panic. And because biological materials are inexpensive and readily available, some thought should be given to whether or not the show will give ideas to potential terrorists. If a person thinks they have been exposed to a biological incident or they suspect a biological threat is planned, they should contact their local health department and/or their local police department. Either of these agencies will promptly notify the FBI, which is responsible for coordinating interagency investigation of bioterrorism. The symptoms for early inhalational anthrax resemble those of the common cold or flu. Anthrax is diagnosed by isolating B. anthracis from the infected person or through other diagnostic tests. The fatality rate for cutaneous anthrax is about 20%; for inhalational anthrax the rate is closer to 75%. Smallpox is spread from person to person via airborne saliva droplets. The majority of people infected with smallpox do recover. There is a fatality rate of approximately 30%.

Case Examples

1. A religious cult has established a base outside town. The cult has applied for a charter to start its own publicly funded school. It's already been voted down once. The charter school is again on the ballot. On election day, hundreds of townspeople become ill and are too sick to get out to vote. In searching the cult headquarters on another matter, a vial of Salmonella typhimurium is found. After questioning by the police, one of the cult members reveals that the salmonella was used to intentionally contaminate local restaurant salad bars with the aim of limiting the number of people who would vote against the charter school.

2. The E.R. is flooded with people experiencing cold and flu symptoms; some people are having difficulty breathing. Police get a phone call from someone claiming they released anthrax in a downtown office building. One of the doctors diagnoses inhalational anthrax. No one on the staff has ever seen a case before. The E.R. is thrown into a panic. Authorities are notified. At shift change, a doctor trained in the Soviet Union comes in and is informed of the case. He has experience with treating anthrax. After viewing a chest x-ray and CT, he's convinced the problem is not anthrax. Cultures come back negative for B. anthracis, proving the doctor was correct in his diagnosis.

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gateway to health communication and social marketing practice

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Bioterrorism - Gateway to Health Communication - CDC

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Human, Social, and Environmental Impacts of Human Genetic Engineering (Maria)

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Human, Social, and Environmental Impacts of Human Genetic Engineering (Maria)

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This article explains the genetic engineering and its effects on many things. It mentions the ethical and religious concerns with the use of genetic engineering. Such as how the process cannot be reversed and what percentage of human genes does an organism have to contain before it is considered human and how many human genes would a green pepper.

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Satyajit Patra*, Araromi Adewale Andrew

American International Medical University, Bosejour Road, Gros Islet, Saint Lucia

Corresponding Author:

Satyajit Patra
American International Medical University
Bosejour Road, Gros Islet, Saint Lucia
E-mail: dr.patra@aimu-edu.us

Citation: Patra S, Andrew AA. Human, Social, and Environmental Impacts of Human Genetic Engineering. J Biomedical Sci. 2015, 4:2. doi:10.4172/2254-609X.100014

Copyright: © 2015 Patra S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Genetic engineering can simply be explained as the alteration of an organism's genetic, or hereditary, material to eliminate undesirable characteristics or to produce desirable new ones. Several works have been done on genetic engineering with major focus on its importance ranging from increasing plant and animal food production, diagnosing disease condition, medical treatment improvement, as well as production of vaccines and other useful drugs. Methods in this techniques involve the selective breeding of animals and plants, hybridization (reproduction between different strains or species), and recombinant deoxyribonucleic acid (rDNA).

Keywords

Genetic Engineering; Ethical and Social Implication

Introduction

Human genetic engineering relies heavily on science and technology. It was developed to help end the spread of diseases. With the advent of genetic engineering, scientists can now change the way genomes are constructed to terminate certain diseases that occur as a result of genetic mutation [ 1]. Today genetic engineering is used in fighting problems such as cystic fibrosis, diabetes, and several other diseases. Another deadly disease now being treated with genetic engineering is the "bubble boy" disease (Severe Combined Immunodeficiency). This is a clear indication that genetic engineering has the potential to improve the quality of life and allow for longer life span [ 2].

Clearly, one of the greatest benefits of this field is the prospect of helping cure illness and diseases in unborn children. Having a genetic screening with a fetus can allow for treatment of the unborn. Overtime this can impact the growing spread of diseases in future generations.

However, these benefits are not without peril. Human genetic engineering is a development that people are either very passionate about or opposed to completely. This article gives a brief account on the effect of this principle on the biosphere together with several controversial issues that accompany the acceptance of this technology [ 3]. The manuscript has been prepared by using information from peer reviewed journals indexed in pubmed in the period of 2000 to 2015.

Effects on the Environment

Although the positive impacts of this field could be enormous, there are many questions raised that needs to be answered. New organisms created by genetic engineering could present an ecological problem. One cannot predict the changes that a genetically engineered species would make on the environment. The release of a new genetically engineered species would also have the possibility of causing an imbalance in the ecology of a region just exotic species would do. An accident or an unknown result could cause several problems. An accident in engineering the genetics of a virus or bacteria for example could result in a stronger type, which could cause a serious epidemic when released. This could be fatal in human genetic engineering creating problems ranging from minor medical problems, to death [ 4].

Effects on Human

Looking at the fact that genetic engineering employs viral vector that carries functional gene inside the human body; the repercussion are still unknown. There are no clues as to where functional genes are being placed. They may even replace the important genes, instead of mutated genes. Thus, this may lead to another health condition or disease to human. Also, as defective genes are replaced with functional gene, then it is expected that there will be a reduction in genetic diversity and if human beings will have identical genomes, the population as a whole will be susceptible to virus or any form of diseases [ 5].

Genetic engineering could also create unknown side effects or outcomes. Certain changes in a plant or animal could cause unpredicted allergic reactions in some people which, in its original form, did not occur. Other changes could result into the toxicity of an organism to humans or other organisms.

Antibiotic Resistance

Genetic engineering often uses genes for antibiotic resistance as "selectable markers." Early in the engineering process, these markers help identify cells that have taken up foreign genes. Although they have no further use, the genes continue to be expressed in plant tissues. Most genetically engineered plant foods carry fully functioning antibiotic-resistance genes.

The presence of antibiotic-resistance genes in foods could have lethal effects. Therefore, eating these foods could reduce the effectiveness of antibiotics to fight disease when these antibiotics are taken with meals. More so, the resistance genes could be transferred to human or animal pathogens, making them impervious to antibiotics. If transfer were to occur, it could aggravate the already serious health problem of antibioticresistant disease organisms [ 6].

Ethical and Social Issues

"Playing God" has become a strong argument against genetic engineering. Several issues have also been raised as regards the acceptance of this technology. These concerns range from ethical issues to lack of knowledge on the effects genetic engineering may have. One major concern is that once an altered gene is placed in an organism, the process cannot be reversed. Public reaction to the use of rDNA in genetic engineering has been mixed. The production of medicines through the use of genetically altered organisms has generally been welcomed. However, critics of rDNA fear that disease-producing, organisms used in some rDNA experiments might develop extremely infectious forms that could cause worldwide epidemics [ 7].

As more human genes are being used in non-human organisms to create new forms of life that are genetically partly human, new ethical questions arise. For instance, what percentage of human genes does an organism have to contain before it is considered human and how many human genes would a green pepper for example have to contain before it can be eaten without qualms. Human genes are now being inserted into tomatoes and peppers to make them grow faster [ 8]. This suggests that one can now be a vegetarian and a cannibal at the same time. For meateaters, the same question could be posed about eating pork with human genes. What about the mice that have been genetically engineered to produce human sperm [ 9]. The question is 'what psychological effect would it pose on the offspring?

Critics have questioned the safety of genetically engineered bovine somatotropin (BST) to increase the milk yield of dairy cows (BST) for both the cows that are injected with it and the humans who drink the resulting milk; owing to the fact that it increases a cow's likelihood of developing mastitis, or infection of the udder, and it also makes cows more susceptible to infertility and lameness [ 6].

Transgenic plants also present controversial issues. Allergens can be transferred from one food crop to another through genetic engineering. Another concern is that pregnant women eating genetically modified products may endanger their offspring by harming normal fetal development and altering gene expression [ 10].

In 2002 the National Academy of Sciences released a report calling for a legal ban on human cloning [ 11]. The report concluded that the high rate of health problems in cloned animals suggests that such an effort in humans would be highly dangerous for the mother and developing embryo and is likely to fail. Beyond safety, the possibility of cloning humans also raises a variety of social issues like the psychological issues that would result for a cloned child who is the identical twin of his or her parent.

Another frightening scenario is the destructive use of genetic engineering. Terrorist groups or armies could develop more powerful biological weaponry. These weapons could be resistant to medicines, or even targeted at people who carry certain genes. Genetically engineered organisms used for biological weapons might also reproduce faster, which would create larger quantities in shorter periods of time, increasing the level of devastation [ 12].

Conclusion

Despite all of these current concerns, the potential for genetic engineering is tremendous. However, further testing and research will be required to educate society on the pros and cons of genetic engineering. There is no doubt that this technology will continue to present intriguing and difficult challenges for 21st century scientists and ethicists, and education and meaningful, respectful discourse are just the starting point of what is required to tackle such complex ethical issues. With the newfound breakthroughs in cloning, the capabilities of changing human characteristics are unpredictable. We can then anticipate intense cross-disciplinary debate and discussion as new life forms are emanating through science and medicine [ 13].

Competing interests

The authors declare that they have no competing interests.

References

1. Patra SAA (2015) Effects of Genetic Engineering - The Ethical and Social Implications. Annals of Clinical and Laboratory Research.

2. Fischer A, Hacein-Bey S, Cavazzana-Calvo M (2002) Gene therapy of severe combined immunodeficiencies. Nat Rev Immunol 2: 615-621.

3. D'Halluin K, Ruiter R (2013) Directed genome engineering for genome optimization. Int J DevBiol 57:621-627.

4. Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA, Flint HJ (1999) Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl Environ Microbiol65:6-10.

5. Jr FW (1996) Viral Genetics. In: Medical Microbiology. 4th Edition edn. University of Texas Medical Branch at Galveston.

6. Mepham TB (2000) The role of food ethics in food policy. The Proceedings of the Nutrition Society 59:609-618.

7. Deuschle K, Fehr M,Hilpert M, Lager I, Lalonde S, et al. (2005) Genetically encoded sensors for metabolites. Cytometry A 64:3-9.

8. Youm JW, Jeon JH, Kim H, Kim YH, Ko K, et al. (2008) Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer's disease. Biotechnollett30: 1839-1845.

9. Naz RK (2009) Development of genetically engineered human sperm immunocontraceptives. J reprodimmunol, 83:145-150.

10. Smith JM (2003) Seeds of Deception: Exposing Industry and Government Lies About the Safety of the Genetically Engineered Foods You're Eating.

11. Powledge TM (2002) Will they throw the bath water out with the baby?: The US Congress is still debating whether to outlaw cloning humans. EMBO Reports 3:209-211.

12. Sayler GS, Ripp S (2000) Field applications of genetically engineered microorganisms for bioremediation processes. Curropin in biotechnol 11:286-289.

13. Powledge TM (2002) Will they throw the bath water out with the baby? The US Congress is still debating whether to outlaw cloning humans. EMBO Rep 3:209-211.

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Human, Social, and Environmental Impacts of Human Genetic Engineering

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Human, Social, and Environmental Impacts of Human Genetic Engineering, Satyajit Patra,Araromi Adewale Andrew

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Stem Cells: The Secret To Change (Azaria)

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Stem Cells: The Secret To Change (Azaria)

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This article explains how there can be different types of stem cells like blood stem cells, embryonic stem cells, and induced pluripotent stem cells. Experts are looking forward to using stem cells to repair of replace many different kinds of tissues for people who were injured in accidents or damaged by diseases.

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Inside your body, red blood cells are constantly on the move. They deliver oxygen to every tissue in every part of your body. These blood cells also cart away waste. So their work is crucial to your survival. But all that squeezing through tiny vessels is tough on red blood cells. That's why they last only about four months.

Where do their replacements come from? Stem cells.

These are a very special family of cells. When most other cells divide, the daughter cells look and act exactly like their parents. For example, a skin cell can't make anything but another skin cell. The same is true for cells in the intestine or liver.

Not stem cells. Stem cells can become many different types. That is how an embryo grows from a single fertilized egg into a fetus with trillions of specialized cells. They need to specialize to make up tissues that function very differently, including those in the brain, skin, muscle and other organs. Later in life, stem cells also can replace worn-out or damaged cells - including red blood cells.

The remarkable abilities of stem cells make them very exciting to scientists. One day, experts hope to use stem cells to repair or replace many different kinds of tissues, whether injured in accidents or damaged by diseases. Such stem cell therapy would allow the body to heal itself. Scientists have found a way to put specialized cells to work repairing damage, too. Together, these cell-based therapies might one day make permanent disabilities a thing of the past.

One unusual type of stem cell offers special promise for such therapeutic uses. For the recent development of this cell type, Shinya Yamanaka shared the 2012 Nobel Prize in medicine.
Meet the family
Blood stem cells live inside your bones, in what is called marrow. There, they divide over and over. Some of the new cells remain stem cells. Others form red blood cells. Still others morph into any of the five types of white blood cells that will fight infections. Although blood stem cells can become any one of these specialized blood cells, they cannot become muscle, nerve or other types of cells. They are too specialized to do that.

Another type of stem cell is more generalized. These can mature into any type of cell in the body. Such stem cells are called pluripotent (PLU ree PO tint). The word means having many possibilities. And it's not hard to understand why these cells have captured the imaginations of many scientists.

Until recently, all pluripotent cells came from embryos. That's why scientists called them embryonic stem cells. After an egg is fertilized, it divides in two. These two cells split again, to become four cells, and so on. In the first few days of this embryo's development, each of its cells is identical to all the others. Yet each cell has the potential to develop into any specialized cell type.

When the human embryo reaches three to five days old, its cells start to realize their potential. They specialize. Some will develop into muscle cells or bone cells. Others will form lung cells - or maybe the cells lining the stomach. Once cells specialize, their "many possibilities" suddenly become limited.

By birth, almost all of a baby's cells will have specialized. Each cell type will have its own distinctive shape and function. For example, muscle cells will be long and able to contract, or shorten. Red blood cells will be small and plate-shaped, so they can slip through blood vessels with ease.

Hidden among all of these specialized cells are pockets of adult stem cells. (Yes, even newborns have "adult" stem cells.) Unlike embryonic stem cells, adult stem cells cannot transform into any and every cell type. However, adult stem cells can replace several different types of specialized cells as they wear out. One type of adult stem cell is found in your marrow, making new blood cells. More types are found in other tissues, including the brain, heart and gut.

Among naturally occurring stem cells, the embryonic type is the most useful. Adult stem cells just aren't as flexible. The adult type also is relatively rare and can be difficult to separate from the tissues in which it is found. Although more versatile, embryonic stem cells are both difficult to obtain and controversial. That's because harvesting them requires destroying an embryo.

Fortunately, recent discoveries in stem cell research now offer scientists a third - and potentially better - option.
The search for answers
In 2006, Shinya Yamanaka discovered that specialized cells - like those in skin - could be converted back into stem cells. Working at Kyoto University in Japan, this doctor and scientist induced - or persuaded - mature cells to become stem cells. He did this by inserting a specific set of genes into the cells. After several weeks, the cells behaved just like embryonic cells. His new type of stem cells are called induced pluripotent stem cells, or iP stem cells (and sometimes iPS cells).

Yamanaka's discovery represented a huge leap forward. The iP stem cells offer several advantages over both embryonic and adult stem cells. First, iP stem cells are able to become any cell type, just as embryonic stem cells can. Second, they can be made from any starting cell type. That means they are easy to obtain. Third, in the future, doctors would be able to treat patients with stem cells created from their own tissues. Such cells would perfectly match the others, genetically. That means the patient's immune system (including all of its white blood cells) would not attack the introduced cells. (The body often mounts a life-threatening attack against transplanted organs that come from other people because they don't offer such a perfect match. To the body, they seem foreign and a potentially dangerous "invader.")

Scientists the world over learned of the technique developed by Yamanaka (who now works at the Gladstone Institutes - which is affiliated with the University of California, San Francisco). Many of these researchers adopted Yamanaka's procedure to create their own induced pluripotent stem cells. For the first time, researchers had a tool that could allow them to make stem cells from people with rare genetic diseases. This helps scientists learn what makes certain cell types die. Experts can also expose small batches of these diseased cells to different medicines. This allows them to test literally thousands of drugs to find out which works best.

And in the future, many experts hope induced stem cells will be used to replace adult stem cells and the cells of tissues that are damaged or dying.
Therapies take patients - and patience
Among those experts is Anne Cherry, a graduate student at Harvard University. Cherry is using induced stem cells to learn more about a very rare genetic disease called Pearson syndrome. A syndrome is a group of symptoms that occur together. One symptom of Pearson syndrome is that stem cells in bone marrow cannot make normal red blood cells. This condition typically leads to an early death.

Cherry has begun to study why these stem cells fail.

She started by taking skin cells from a girl with the disease. She placed the cells in a test tube and added genes to turn them into stem cells. Over several weeks, the cells began to make proteins for which the inserted genes had provided instructions. Proteins do most of the work inside cells. These proteins turned off the genes that made the cells act like skin cells. Before long, the proteins turned on the genes to make these cells behave like embryonic stem cells.

After about three months, Cherry had a big batch of the new induced stem cells. Those cells now live in Petri dishes in her lab, where they are kept at body temperature (37° Celsius, or 98.6° Fahrenheit). The scientist is now trying to coax the induced stem cells into becoming blood cells. After that, Cherry wants to find out how Pearson syndrome kills them.

Meanwhile, the patient who donated the skin cells remains unable to make blood cells on her own. So doctors must give her regular transfusions of blood from a donor. Though life-saving, transfusions come with risks, particularly for someone with a serious disease.

Cherry hopes to one day turn the girl's induced stem cells into healthy new blood stem cells - and then return them to the girl's body. Doing so could eliminate the need for further transfusions. And since the cells would be the girl's own, there would be no risk of her immune system reacting to them as though they were foreign.
Sight for sore eyes
At University of Nebraska Medical Center in Omaha, Iqbal Ahmad is working on using stem cells to restore sight to the blind. A neuroscientist - someone who studies the brain and nervous system - Ahmad has been focusing on people who lost sight when nerve cells in the eye's retina died from a disease called glaucoma (glaw KOH muh).

Located inside the back of the eye, the retina converts incoming light into electrical signals that are then sent to the brain. Ahmad is studying how to replace dead retina cells with new ones formed from induced pluripotent stem cells.

The neuroscientist starts by removing adult stem cells from the cornea, or the clear tissue that covers the front of the eye. These stem cells normally replace cells lost through the wear and tear of blinking. They cannot become nerve cells - at least not on their own. Ahmad, however, can transform these cells into iP stem cells. Then, with prodding, he turns them into nerve cells.

To make the transformation, Ahmad places the cornea cells on one side of a Petri dish. He then places embryonic stem cells on the other side. A meshlike membrane separates the two types of cells so they can't mix. But even though they can't touch, they do communicate.

Cells constantly send out chemical signals to which other cells respond. When the embryonic stem cells "speak," the eye cells "listen." Their chemical messages persuade the eye cells to turn off the genes that tell them to be cornea cells. Over time, the eye cells become stem cells that can give rise to different types of cells, including nerve cells.

When Ahmad's team implanted the nerve cells into the eyes of laboratory mice and rats, they migrated to the retina. There, they began replacing the nerve cells that had died from glaucoma. One day, the same procedure may restore vision to people who have lost their sight.
Another approach
In using a body's own cells to repair injury or to treat disease, stem cells aren't always the answer. Although stem cells offer tremendous advances in regenerating lost tissue, some medical treatments may work better without them. That's thanks to the chemical communication going on between all cells all of the time. In some situations, highly specialized cells can act as a conductor, directing other cells to change their tune.

In 2008, while working at the University of Cambridge in England, veterinary neurologist Nick Jeffery began a project that used cells taken from the back of the nose. But Jeffery and his team were not out to create stem cells. Instead, the scientists used those nasal cells to repair damaged connections in the spinal cord.

The spinal cord is basically a rope of nerve cells that ferry signals to and from the brain and other parts of the body. Injuring the spinal cord can lead to paralysis, or the loss of sensation and the inability to move muscles.

Like Ahmad, some researchers are using stem cells to replace damaged nerve cells. But Jeffery, now at Iowa State University in Ames, doesn't think such techniques are always necessary to aid recovery from spinal injuries. "Stem cell transplantation," points out Jeffery's colleague, neuroscientist Robin Franklin, "is to replace a missing cell type." In a spinal injury, the nerve cells aren't missing. They're just cut off.

Nerve cells contain long, wirelike projections - called axons - that relay signals to the next cell. When the spine is injured, these axons can become severed, or cut. Damaging an axon is like snipping a wire - the signal stops flowing. So the Cambridge scientists set out to see if they could restore those signals.

Jeffery and his fellow scientists work with dogs that have experienced spinal injuries. Such problems are common in some breeds, including dachshunds. The team first surgically removed cells from the dogs' sinuses - or the hollow spaces in the skull behind the nose. These are not stem cells. These particular cells instead encourage nerve cells in the nose to grow new axons. These cells help the pooches maintain their healthy sense of smell.

The scientists grew these sinus cells in the lab until they had reproduced to large numbers. Then the researchers injected the cells into the spinal cords of two out of every three doggy patients. Each treated dog received an injection of its own cells. The other dogs got an injection of only the liquid broth used to feed the growing cells.

Over several months, the dogs' owners repeatedly brought their pets back to the lab for testing on a treadmill. This allowed the scientists to evaluate how well the animals coordinated their front and hind feet while walking. Dogs that had received the nasal cells steadily improved over time. Dogs that received only the liquid did not.

This treatment did not result in a perfect cure. Nerve cells did reconnect several portions of the spinal cord. But nerve cells that once linked to the brain remained disconnected. Still, these dog data indicate that nasal cells can aid in recovering from a spinal cord injury.

Such new developments in cellular research suggest that even more remarkable medical advancements may be just a few years away. Yamanaka, Cherry, Ahmad, Jeffery, Franklin and many other scientists are steadily unlocking secrets to cellular change. And while you can't teach an old dog new tricks, scientists are finding out that the same just isn't true of cells anymore.

cornea The clear covering over the front of the eye.

embryo A vertebrate, or animal with a backbone, in its early stages of development.

gene A section of DNA that carries the genetic instructions for making a protein. Proteins do most of the work in cells.

glaucoma An eye disease that damages nerve cells carrying signals to the brain.

immune cell White blood cell that helps protect the body against germs.

molecule A collection of atoms.

neuron (or nerve cell) The basic working unit of the nervous system. These cells relay nerve signals.

neuroscientist A researcher who studies neurons and the nervous system.

paralysis Loss of feeling in some part of the body and an inability to move that part.

retina The light-sensitive lining at the back of the eye. It converts light into electrical impulses that relay information to the brain.

sinus An opening in the bone of the skull connected to the nostrils.

spinal cord The ropelike collection of neurons that connect the brain with nerves throughout the body.

tissue A large collection of related, similar cells that together work as a unit to perform a particular function in living organisms. Different organs of the human body, for instance, often are made from many different types of tissues. And brain tissue will be very different from bone or heart tissue.

transfusion The process of transferring blood into one person that had been collected from another.
Word Find (click here to enlarge puzzle for printing)

Alison Pearce Stevens is a former biologist and forever science geek who writes about science and nature for kids. She lives with her husband, their two kids and a small menagerie of cuddly (and not-so cuddly) critters.

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Stem cells: The secret to change | Science News for Students

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Inside your body, red blood cells are constantly on the move. They deliver oxygen to every tissue in every part of your body. These blood cells also cart away waste. So their work is crucial to your survival. But all that squeezing through tiny vessels is tough on red blood cells.

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Using chickens eggs, to practice with cloning stem cells

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Using chickens eggs, to practice with cloning stem cells

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This article explains how scientists are slowly becoming able to produce a clone of a chickens egg. This procedure is being on by Crisper. The scientists use Primordial germ cells, also known as (PG'C). This ties into the sperm of the egg that is altered by scientists.

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Abstract

Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.

Introduction

The chicken is a useful animal model for biological research and can be used to produce biopharmaceutical products that cannot be produced in mammalian bioreactor systems1,2. Chicken meat and eggs derived from 70 billion chickens yearly are an important source of high quality protein, vitamins and minerals in the global economy3. The ability to precisely edit the chicken genome to introduce or test genetic variants will aid the study of gene function, define combinatorial allelic contribution to disease resistance/resilience and production phenotypes and will lead to the uncovering of beneficial alleles which could be introduced into breeding programmes for the improvement of poultry welfare and sustainable production.

The application of precision genome editing to bird species has failed to keep pace with that of other mammalian species. In mammals, germline genetic engineering may be achieved through pronuclear injection of the zygote4, injection of genetically modified embryonic stem cells into the blastocyst5, and somatic cell nuclear transfer of genetically modified somatic cells (SCNT)6,7. These methods are not regularly practised in bird species because the single-cell zygote is difficult to access and manipulate and cultured avian embryonic stem cells do not contribute to the formation of the germ lineage8,9. In contrast to other species, heritable genetic changes may be introduced into chicken through the genetic manipulation of primordial germ cells (PGCs), the stem cell precursors of the sperm and egg, which can be propagated in culture and will contribute to the germline when reintroduced into surrogate host chick embryos.

Targeted genetic modification in chicken PGCs was first demonstrated through classical gene targeting by homologous recombination (HR) to generate immunoglobulin-knockout chickens13. The observation that DNA Double-stranded breaks (DSBs) stimulate and increase the frequency of HR led to the development of artificial site-specific nucleases including ZFNs, TALENs and CRISPR/Cas9 with the goal of improving the efficiency of site-specific gene targeting14,15. Artificial site-specific nucleases are guided to a specific genomic site by programmable DNA-binding modules where they create a DSB. The cleaved DNA is immediately repaired by either non-homologous end-joining (NHEJ) or the HR pathway. NHEJ is the predominant pathway that repairs DSBs that occur in all phases of the cell cycle and often leads to the generation of insertion/deletion (INDEL) mutations19. The HR or homology-directed repair (HDR) pathway is active in the S and G2 phase of the cell cycle and is used to repair a double-stranded DNA break when there is an available DNA donor containing a region that is homologous to the region surrounding the severed DNA ends. The high fidelity of HDR is constrained by the nucleotide composition of the repair template and this constraint is exploited in genome engineering to introduce a desired nucleotide change. TALENs and CRISPR/Cas9-mediated NHEJ have been used to produce several knockout-chickens through the generation of INDELs in chicken PGCs22,23. Homologous recombination mediated by TALENs and CRISPR/Cas9 have also been performed in chicken PGCs to introduce targeted transgenes24,25. However, the use of site-specific nucleases to perform precision editing of a single to few nucleotides has not been demonstrated in avian species.

Genome editing mediated by CRISPR/Cas9 and short single stranded oligodeoxynucleotides (ssODN) donors has been used to perform small precise genetic changes in many cell types and organisms26,27,28,29,30,31,32,33,34,35,36,37 and in a chicken somatic cell line38. However, use of ssODN donors for gene correction can be toxic to cells by causing a G2/M cell cycle arrest39, and activating cellular immune responses40. HDR targeting therefore requires careful optimization for each cell type. Following CRISPR/Cas9-induced DSBs, DNA repair with ssODN donors occurs through the synthesis-dependent strand annealing (SDSA) pathway of HDR41. However, the accuracy of HDR editing may be distorted by the incorporation of INDELs at the target site in a second round of repair due to re-cleaving by CRISPR/Cas942,43. Previous studies have shown that the introduction of Cas9-blocking mutations in the PAM are effective in preventing re-editing of genetic loci while blocking mutations positioned in the gRNA target sequence have variable efficacy35.

High fidelity CRISPR/Cas9 nucleases with improved specificity have been developed to reduce the frequency of off-target events associated with wild type Streptococcus pyogenes Cas9 (SpCas9-WT). These high fidelity Cas9 variants harbour amino acid substitutions that significantly reduce activation of cleavage at target sites that are not perfectly complementary to the gRNA sequence. Here, we investigated a high fidelity Cas9 variant, SpCas9-HF145, for introducing defined nucleotide changes in chicken PGCs using ssODN donors. First, we optimised the use of ssODN donors as repair templates for CRISPR/Cas9-mediated HDR in cultured chicken PGCs. We then directly compared HDR editing between SpCas9-WT and SpCas9-HF1 using ssODN donors containing CRISPR/Cas9-blocking mutations positioned in the PAM and show that many loci in chicken PGCs can be efficiently edited using SpCas9-HF1. Using ssODN donors containing mutations in the guide sequence only, we also showed that SpCas9-HF1 is more efficient than SpCas9-WT in introducing precise genome edits in the absence of CRISPR/Cas9-blocking mutation in the PAM. We subsequently used a eGFP-to-BFP conversion assay47 to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and found that SpCas9-HF1 increases the efficiency of accurate HDR editing while reducing INDEL formation at the target site. Finally, we combined SpCas9-HF1 and ssODN donors to demonstrate precise biallelic and monoallelic introduction of the chicken scaleless genetic variant associated with the heat tolerance featherless phenotype by introducing two defined nucleotide substitutions into the FGF20 gene. Our results demonstrate the use of high fidelity CRISPR/Cas9 variants to perform precise HDR genome editing with high efficiency in chicken PGCs.

Results

High fidelity Cas9 variant, SpCas9-HF1, shows efficient HDR editing in chicken PGCs

We first tested SpCas9-WT and high fidelity SpCas9-HF1 to edit multiple loci in chicken PGCs. SpCas9-HF1 contains 4 amino acid substitutions that prevent activation of the nuclease at mismatched targets45. To directly compare SpCas9-HF1 and SpCas9-WT, we transferred the codon-changing mutations from the VP12 vector which encodes SpCas9-HF1 into PX459 vector which encodes a mammalian-codon optimised SpCas9-WT as well as puromycin resistance33 (Fig. 1). We named this modified vector HF-PX459.

Figure 1

Schematic representation of CRISPR/Cas9 vectors used in this study. HF-PX459 and PX459 vectors differ by only four amino acids in the encoded Cas9 protein. HF-PX459 vector encodes SpCas9-HF1 while PX459 encodes SpCas9-WT. The U6 promoter drives sgRNA expression while expression of Cas9 protein and puromycin resistance (PuroR) is driven by the CAG promoter.

To compare HDR editing between SpCas9-WT and SpCas9-HF1 in chicken PGCs, we designed gRNAs to target exon 3 of FGF20 ( FGF20-gRNA1) and exon 2 of CXCR4 ( CXCR4-gRNA), and used previously described gRNAs to target exon 3 of ovalbumin ( OVA) and exon 1 of ovomucoid ( OVM) ( OVA-gRNA for ovalbumin and OVM-gRNA for ovomucoid)23 (Fig. 2). In order to directly analyse HDR efficiency without sequencing PCR products, we used antisense repair templates that introduce an EcoRI recognition site for RFLP analysis51,52. CXCR4 is expressed in chicken PGCs while FGF20, ovalbumin and ovomucoid are transcriptionally inactive. To target each locus, we used ssODN donors containing mutations of the gRNA seed sequence and PAM to insert an EcoRI recognition sequence (122-nt CXCR4-ssODN for CXCR4, 126-nt FGF20-ssODN for FGF20, 127-nt OVA-ssODN for ovalbumin and 128-nt OVM-ssODN for ovomucoid; Fig. 2). We co-transfected the corresponding ssODN donor and gRNA with SpCas9-HF1 or SpCas9-WT into PGCs and then treated with puromycin to select for Cas9-transfected cells. We performed two independent targeting experiments for each locus. To analyse HDR, we PCR amplified the target site and performed EcoRI RFLP digest assay on the PCR products to estimate HDR efficiency. In CXCR4, we observed an average HDR efficiency of 50.5% in PGCs targeted with SpCas9-HF1 and 35.5% with SpCas9-WT (Fig. 2A and Supplementary Fig. S2). For ovomucoid, the average HDR efficiency was 42.5% using SpCas9-HF1 and 39.5% with SpCas9-WT (Fig. 2B and Supplementary Fig. S2), whereas the average HDR efficiency was 63.5% with SpCas9-HF1 and 62.5% with SpCas9-WT for ovalbumin (Fig. 2C and Supplementary Fig. S2). For FGF20, we also tested the NHEJ inhibitors, SCR7 and L755507, reported to increase HDR efficiency34,53. We observed an average HDR efficiency of 48.5% and 3.5% with SpCas9-HF1 and SpCas9-WT respectively without using NHEJ inhibitors while no HDR improvement was observed with either SCR7 or L755507 treatment (Fig. 2D and Supplementary Fig. S2). Thus, NHEJ inhibitors do not increase targeting efficiency in PGCs and targeting efficiencies were equal or slightly better using SpCas9-HF1 in combination with donor containing PAM mutations.

Figure 2

Efficient oligonucleotide-template HDR editing across multiple loci using SpCas9-HF1. Left panels: PAM-region mutated templates. Right panels: Guide-region mutated templates. All HDR ssODN donors were complementary to the gRNA target strand and were symmetric around the cut site except for FGF20-ssODN. gRNA sequences are highlighted in blue. PAMs are highlighted in purple. Blue arrowheads indicate Cas9 cleavage sites. Insertions, deletions and substitutions in HDR donors are highlighted in red. Black arrowheads indicate undigested PCR substrate. Red arrowheads indicate digested PCR products. ( −) untreated substrate. (+) treated substrate. (See Figure S9 for uncropped images).

We next tested whether introducing single-nucleotide blocking mutations into the PAM-distal region of the gRNA target sequence was sufficient to achieve high HDR without introducing a blocking mutation in the PAM. Cleavage activity of Cas9 was shown to be severely reduced when mismatches are present in the seed region (first 8-12 nucleotides proximal of the PAM) of the gRNA target sequence54,55,56,57. However, mismatches were tolerated in the PAM-distal non-seed region of the gRNA target and are associated with off-target mutagenesis by SpCas9-WT. To test the efficiency of accurate HDR when introducing single nucleotide mutations into the non-seed region of the gRNA target sequence, we designed ssODN donors containing one to three substitutions of the last 14-20 PAM-distal nucleotides (120-nt CXCR4-ssODN2 for CXCR4, 128-nt OVM-ssODN2 for ovomucoid, 127-nt OVA-ssODN2 for ovalbumin, 127-nt OVA-ssODN3 for ovalbumin and 126-nt FGF20-ssODN2 for FGF20; Fig. 2, right panels). The substitutions introduce a restriction site for RFLP analysis. We transfected and analysed cells as above. For CXCR4, we observed an average HDR efficiency of 17.5% using SpCas9-HF1 and 0.0% with SpCas9-WT when CXCR4-ssODN2 was supplied as repair template (Fig. 2A and Supplementary Fig. S2). For ovomucoid, the average HDR efficiency was 34.0% using SpCas9-HF1 and 0.5% with SpCas9-WT when OVM-ssODN2 was supplied as repair template (Fig. 2B and Supplementary Fig. S2). For ovalbumin, the average HDR efficiency was 58.5% using SpCas9-HF1 and 53.5% using SpCas9-WT when OVA-ssODN2 was supplied as repair template (Fig. 2C and Supplementary Fig. S2). When OVA-ssODN3 was used as a repair template containing a single base pair change in the PAM distal guide region, we observed an average HDR efficiency of 43.5% using SpCas9-HF1 and 1.0% using SpCas9-WT (Fig. 2C and Supplementary Fig. S2). For FGF20, we observed an average HDR efficiency of 37.5% with SpCas9-HF1 and 3.5% with SpCas9-WT when FGF20-ssODN2 was supplied as repair template (Fig. 2D and Supplementary Fig. S2). We also compared symmetrical and asymmetrical ssODNs as well as a double stranded repair template carried in plasmid but observed similar levels of HDR at FGF20 (data not shown). Our results show that SpCas9-HF1, in comparison to SpCas9-WT, is effective for achieving precise introduction of single nucleotide changes into the non-seed region of the gRNA target sequence. These results demonstrate the accuracy and versatility of SpCas9-HF1.

SpCas9-HF1 reduces INDEL formation at target site in comparison to SpCas9-WT

To better quantitate HDR and INDEL formation mediated by SpCas9-WT and SpCas9-HF1, we used an assay that converts enhanced green fluorescent protein ( eGFP) to blue fluorescent protein ( BFP) after editing events47. The eGFP-to-BFP conversion assay simultaneously quantifies total HDR and NHEJ events in a targeted population. We targeted PGCs isolated from homozygous transgenic chicken ubiquitously and constitutively expressing eGFP (GFP-PGCs; Fig. 3A) with a validated gRNA ( GFP-gRNA) which was co-delivered with SpCas9-WT or SpCas9-HF1 and a ssODN donor carrying three nucleotide substitutions ( BFP-ssODN; Fig. 3A) designed to convert eGFP to BFP47. In this case the 20 nucleotide GFP-gRNA begins with a C nucleotide which reduces transcription from the U6 promoter61. BFP-ssODN donor contains a C-to-G substitution that converts Threonine (T) to Serine (S), a T-to-C substitution that converts Tyrosine (Y) to Histidine (H) and a synonymous T-to-G substitution. The C-to-G substitution (the 1 st nucleotide of the gRNA seed sequence) and the T-to-C substitution (1 st nucleotide of the PAM) serve as Cas9-blocking mutations to prevent re-editing and increase HDR accuracy of SpCas9-WT. eGFP is converted to BFP by a Y66H amino acid substitution. Error-free editing of the eGFP sequence will lead to the expression of BFP while the presence of INDELs, even after recombinational repair, will result in no BFP or eGFP expression due to a shift in the reading frame. Before transfection, 99.9% of gated living cells expressed eGFP. Transfection with SpCas9-WT vector resulted in 30.3% of PGCs expressing BFP and the remaining 69.7% did not express GFP indicating INDEL formation. In contrast, transfection with SpCas9-HF1 vector resulted in 68.2% of PGCs expressing BFP and the remaining 31.7% did not express eGFP (Fig. 3B). The HDR and INDEL levels obtained with SpCas9-WT in this assay are consistent with reports by other researchers using human cell lines47,62. Two distinct populations of BFP-expressing PGCs were observed for both SpCas9-WT and SpCas9-HF1 (Fig. 3C, top panel) and determined their median fluorescent intensity (MFI). We determined that the population with the lower MFI of approximately 2000 units was monoallelic for BFP and contained INDELs on the second GFP allele while the population with an MFI of approximately 4000 units was biallelic for BFP by TIDE analysis63 of the PCR sequencing traces of these populations (Fig. 3C, bottom panel, Supplementary Fig. S3). Interestingly, we noticed that the proportion of BFP PGCs transfected with SpCas9-HF1 that was biallelic for BFP was 53.9% while 44.7% was monoallelic for BFP. For PGCs transfected with SpCas9-WT, 25.4% of BFP PGCs was biallelic for BFP while 73.6% was monoallelic for BFP. This indicates that SpCas9-HF1 increases the efficiency of biallelic HDR by up to two-fold by reducing INDEL formation. Also, the absence of PGCs expressing only eGFP or co-expressing eGFP and BFP is indicative of the high mutagenic activity of CRISPR/Cas9 and is similar to previous observation47,62. Our results show that using SpCas9-HF1 with ssODN donors containing Cas9-blocking mutations positioned in the gRNA sequence increases HDR levels by more than 2-fold with a concomitant decrease in INDEL formation in comparison to SpCas9-WT.

Figure 3

SpCas9-HF1 increases HDR by reducing INDEL formation. ( A) An eGFP expression cassette was integrated in the MAD1L1 gene. CAG promoter drives eGFP expression. The gRNA sequence is highlighted in blue while the PAM is highlighted in purple. Blue arrowhead indicates Cas9 cleavage site. Substitutions in the BFP-ssODN donor are highlighted in red. BFP-ssODN is a 122-nt HDR template that is complementary to the target strand. ( B) Flow cytometric detection of BFP-expressing PGCs and non-fluorescent PGCs to quantify HDR and INDEL levels respectively. ( C) Flow cytometric quantification of BFP-expression PGCs (top panels) and mean fluorescent intensity of BFP-expressing PGCs (bottom panels). ( D) Alignment of sequences of cloned PCR products from PGCs in quadrants Q1 and Q4.

SpCas9-HF1 efficiently introduces heterozygous biallelic edits in comparison to SpCas9-WT

In the experiment above (Fig. 3), we observed that less than 0.2% of PGCs were heterozygous for eGFP and BFP using SpCas9-HF1 while no BFP-eGFP heterozygotes were obtained using SpCas9-WT. This observation reflects the experimental difficulty in generating specific heterozygous mutations since most CRISPR/Cas9 editing events are biallelic and cells with monoallelic HDR edits usually contain INDELs on the second allele35,64. A strategy that has been employed for editing single alleles in human IPS cells uses a mixture of two ssODN donor templates containing Cas9-blocking mutations with an observed efficiency of 0.1%35. Since SpCas9-HF1 increases HDR levels as well as the efficiency of biallelic HDR by reducing INDEL formation (Fig. 3B and C), we reasoned that SpCas9-HF1 could increase the efficiency of editing individual alleles using two ssODN donors. We compared SpCas9-WT and SpCas9-HF1 by performing eGFP-to-BFP editing of single eGFP alleles in GFP-PGCs to produce eGFP/BFP heterozygote cells. We designed a second repair template ( GFP-ssODN) containing three synonymous nucleotide substitutions to preserve the amino acid sequence of eGFP (Fig. 4A). GFP-PGCs were then transfected with SpCas9-WT or SpCas9-HF1 vectors and equimolar amounts of GFP-ssODN and BFP-ssODN donors and then analysed by flow cytometry for expression of BFP and eGFP. The results from two independent experiments are shown in Fig. 4B. 1.5% of PGCs targeted with SpCas9-WT co-expressed eGFP and BFP while 9.2% of PGCs targeted with SpCas9-HF1 were eGFP/BFP co-expressing cells reflecting an almost 7-fold increase in HDR frequency in comparison to SpCas9-WT. Direct sequencing of PCR products from single-cell clones co-expressing eGFP and BFP confirmed incorporation of nucleotide changes in ssODN donors into the individual eGFP alleles (Fig. 4C). Similar to our previous result (Fig. 3B), we observed that 74.4% of PGCs targeted with SpCas9-WT did not express eGFP or BFP in comparison to 31.8% for PGCs targeted with SpCas9-HF1 (Fig. 4B). These results illustrate that SpCas9-HF1 increases the frequency of editing individual alleles by increasing HDR efficiency while reducing INDEL formation.

Figure 4

SpCas9-HF1 increases the frequency of single allele editing ( A) GFP-ssODN contains three synonymous substitutions to preserve that amino acid sequence of eGFP. Substitutions in ssODN donors are highlighted in red. gRNA target sequence is coloured blue, PAM is highlighted in purple, blue arrowhead indicates Cas9 cleavage site. ( B) Flow cytometric detection and quantification of PGCs co-expressing GFP and BFP. ( C) Sanger sequencing traces of direct PCR products from representative isolated single cell clones co-expressing GFP and BFP. Red box encloses region containing edits.

Precise biallelic introgression of a genetic variant into chicken PGCs

We next demonstrated the introgression of specific genetic variants into PGCs. The scaleless mutation ( sc/sc) is a single A-to-T substitution in exon 3 of FGF20 (535A > T) which creates a premature stop codon resulting in a truncated FGF20 protein that leads to a complete loss of feather development48. We selected three gRNAs (Supplementary Fig. S1) targeting for the location of the scaleless variant but only gRNA1 ( FGF20-gRNA1) containing a cut site that is 12 bp away from the target nucleotide was active with both SpCas9-WT and SpCas9-HF1 (Fig. 5A and Supplementary Fig. S1). We designed an ssODN donor ( Sca-ssODN) containing a Cas9-blocking synonymous point mutation in the PAM (AGG → AGA) and the scaleless mutation (535A > T) which was 6 bp downstream of the 3′ end of the PAM (Fig. 5A). We anticipated that the 12 bp distance from the cut site to the edit site (cut-to-edit distance) would reduce editing accuracy. It has previously been shown that the efficiency of heterologous DNA incorporation is reduced as the distance between the site of edit and Cas9 cleavage site increases and the highest efficiency is achived within a distance of 8 to 10 bp. To address the cut-to-edit distance, we used an asymmetric design for the ssODN donor containing a left homology arm (HA) of 36 bp and right HA of 91 bp to provide increased homology on the side containing the edited PAM and the 535A >  T mutation. This asymmetric repair template design was previously described to increase HDR efficiency in human HEK293 and K562 cell lines by up to 60%66.

Figure 5

Introgression of scaleless 535 A > T mutation into FGF20. ( A) Strategy for introduction of 535 A > T mutation into Exon 3. The gRNA directs cleavage 12 bp from the position of the targeted nucleotide change. Substitutions in ssODN donors are highlighted in red. PAM is highlighted in purple. Blue arrowhead indicates Cas9 cleavage site. All ssODN donors are complementary to the target strand. ( B- D) Frequency of the mutation genotypes of isolated single-cell clones based on ssODN donor used for allelic repair. Representative sequence traces show mutation genotypes of HDR clones. Black arrows indicate PAM mutations, red arrows indicate Sc mutations, blue arrow shows biallelic 535 A  > C mutation.

To introduce the 535A >  T ( sc/sc) gene variant, we transfected PGCs with FGF20-gRNA1 and SpCas9-HF1 or SpCas9-WT with Sca-ssODN and then sequenced PCR products directly from 38 single-cell clonal populations isolated from two independent experiments to analyse their mutational status and zygosity. The results are shown in Table 1 and Fig. 5B.

Table 1 Edits of the FGF20 gene based on ssODN donor used for allelic repair in single-cell clones targeted with Sca-ssODN and SpCas9 or SpCas9-HF1 (Supplementary Figs S4 and S5).

We found that 7.9% of isolated clones transfected with SpCas9-WT contained precise monoallelic introduction of the PAM substitution and the scaleless mutation on one chromosome while the other chromosome contained INDELs. The other 92.1% of isolated clones contained INDELs on both chromosomes with no incorporation of the scaleless mutation. We note that this frequency is much higher than the INDEL frequency measured for this guide using the T7 endonuclease I assay (Fig. S1). We attribute this difference to inefficiencies of the T7 endonuclease I assay67. In contrast, 41.8% of single-cell clones transfected with SpCas9-HF1 were precise biallelic HDR clones. We discovered that 36.8% of the SpCas9-HF1 clones contained accurate biallelic incorporation of the PAM substitution and the 535A >  T ( sc/sc) mutation (Fig. 5B). We also noted that the PAM and 535A >  T mutation was incorporated into only one chromosome ( sc/+) in two SpCas9-HF1 clones (5.3% of isolated clones), while the second chromosome only contained the PAM substitution. Furthermore, we observed that 18.4% of the total SpCas9-HF1 clones were precise monoallelic HDR clones containing the PAM substitution and 535A >  T mutation on one chromosome while the other chromosome contained INDELs. The high rate of biallelic editing is similar to observations in human IPSCs and mouse ES cells targeted with SpCas9-WT35,64. We noted that the 535A >  T mutation was incorporated into 37 out of 39 HDR alleles (94.9%) in SpCas9-HF1 clones.

We next attempted to introduce the 535A >  T gene variant into a single allele ( sc/+) by providing two repair templates. Our previous result (Fig. 4B) showed that SpCas9-HF1 increases the overall efficiency of editing individual alleles using two ssODN donors whereas isolation of biallelically edited heterozygotes was barely detectable when SpCas9 was used. Consequently, we only tested SpCas9-HF1 in the following experiment. We designed two donors to introduce silent mutations on one allele while incorporating the 535 A >  T mutation into the second allele using Sca-ssODN (Fig. 5A). The first silent donor (Silent-ssODN) contained 2 synonymous substitutions in the PAM (AGG → CGC) to preserve the FGF20 amino acid sequence. In the second silent donor (Silent2-ssODN), the PAM mutation was the same as in Sca-ssODN (AGG → AGA) while a synonymous substitution 535 A >  C was made in the same position as the 535 A >  T mutation 12 bp from the cleavage site to maintain the cut-to-edit distance between templates. Since Silent-ssODN showed more complementarity to the target region than Sca-ssODN due to the 535 A >  T substitution, we asked whether the two silent repair templates would be used at different frequencies for allelic repair when used with Sca-ssODN.

We transfected SpCas9-HF1 with FGF20-gRNA1 and an equimolar mixture of Sca-ssODN donor and Silent-ssODN (Sca-ssODN/Silent-ssODN mixture) or an equimolar mixture of Sca-ssODN and Silent2-ssODN (Sca-ssODN/Silent2-ssODN mixture) for comparison. We performed two independent experiments and isolated a total of 18 single-cell clonal populations in each experiment. The results are shown in Tables 2 and 3 and Fig. 5C,D.

Table 2 Edits of the FGF20 gene based on ssODN donor used for allelic repair in single-cell clones targeted with SpCas9-HF1 and a mixture of Sca-ssODN and Silent-ssODN (Supplementary Fig. S6).

Table 3 Edits of FGF20 gene based on ssODN donor used for allelic repair single-cell clones targeted with SpCas9-HF1 and a mixture of Sca-ssODN and Silent2-ssODN (Supplementary Fig. S7).

Biallelic HDR editing with the co-incorporation of PAM mutations into the two alleles was observed in 69.5% of the clones targeted with the Sca-ssODN/ Silent-ssODN mixture in contrast to 55.5% observed with the Sca-ssODN/ Silent2-ssODN mixture (Fig. 5C). Remarkably, 25% of the clones contained the heterozygous edit for 535 A >  T ( sc/+) in which the two alleles were independently repaired by the two ssODN templates ( Sca/Silent and Sca/Silent2; Tables 2 and 3). 13.9% of the isolated clones targeted with the Sca-ssODN/ Silent-ssODN mixture were biallelically repaired by Sca-ssODN ( Sca/Sca) whereas 25.0% of the clones were biallelically repaired by Silent-ssODN ( Silent/Silent) (Table 2 and Fig. 5C). One clone ( Sca/Sca*Wt) was biallelically repaired by Sca-ssODN and was homozygous for the PAM substitution but heterozygous for 535 A >  T ( sc/+) indicating partial introduction of edits in one chromosome (Table 2). We noted that 30 out of the 53 HDR alleles (56.6%) were repaired by Silent-ssODN when Sca-ssODN and Silent-ssODN were used together. Similarly, Silent2-ssODN repaired 57.7% of the 52 HDR alleles generated when it was used with Sca-ssODN. Using Sca-ssODN and Silent2-ssODN together, we also observed that 22.2% of the total isolated clones were biallelically repaired by Silent2-ssODN ( Silent2/Silent2) while 13.9% were biallelically repaired by Sca-ssODN ( Sca/Sca and Sca/Sca*Wt). We identified 2 clones biallelically harbouring the AGG → AGA PAM substitution on the two alleles but containing 535 A >  C mutation on only one allele indicating partial introduction of edits. While the proportion of biallelically edited heterozygous clones were the same using the two ssODN donor mixtures (Sca-ssODN/Silent-ssODN and Sca-ssODN/Silent2-ssODN mixtures), we observed that clones with monoallelic HDR contained INDELs on the other allele. The 535 A >  T substitution was incorporated into 22 out of 23 HDR alleles (95.7%) repaired by Sca-ssODN in clones targeted with Sca-ssODN/ Silent-ssODN mixture. Similarly, the 535 A >  T substitution was incorporated into all 22 HDR alleles (100.0%) repaired by Sca-ssODN in clones targeted with Sca-ssODN/ Silent2-ssODN mixture.

Discussion

The validation of many genotypes requires the accurate creation of specific biallelic or monoallelic combinations by the introduction of single to several nucleotide changes. Building on previous work, our results illustrate an efficient strategy for introducing defined sequence changes into PGCs using the CRISPR/Cas9 system. We show that ssODNs serve as efficient donors for precision genome editing in chicken PGCs. To the best of our knowledge, this has not been previously demonstrated for avian species or for germline stem cells. Following CRISPR/Cas9-induced DSBs, DNA repair with ssODN donors occurs through the synthesis-dependent strand annealing (SDSA) pathway41. We observe that HDR efficiencies with ssODN donors in chicken PGCs are up to 5-fold higher than previously reported using double stranded templates24,25. Previous reports show that Cas9 activity may be repressed in transcriptionally inactive targets in heterochromatin and nucleosomal DNA. We found that HDR efficiency was unaffected by the transcriptional state of the targeted gene in PGCs (Fig. 2).

Similar to a recent report in human cells71, we observed using the GFP-to-BFP conversion assay that SpCas9-HF1 increases HDR levels while reducing INDEL formation (Figs 3 and 4). Since enhanced specificity Cas9 variants discriminate against targets bearing mismatches in the non-seed region of the gRNA target and prevent nuclease activation, the higher HDR efficiency observed with SpCas9-HF1 in our results can be attributed to the high fidelity of the nuclease which proof-reads the gRNA target sequence before activating cleavage, thereby reducing re-editing of the repaired target site and leading to higher levels of HDR in the two alleles and lowering INDEL formation. As a consequence, base pair changes can be efficiently introduced into the non-seed sequence of the guide region using SpCas9-HF1 without introducing a blocking mutation into the neighbouring PAM site (Fig. 2). This enhancement in HDR accuracy by SpCas9-HF1 directly increases the efficiency of editing single alleles to generate PGCs with specific heterozygous genotypes (Figs 4 and 5C,D).

In human IPS cells, use of SpCas9-WT and ssODN donors containing appropriate CRISPR/Cas9-blocking mutations positioned in the PAM site increased HDR levels by up to a 100-fold while mutations positioned in the gRNA target sequence showed variable efficacy35. At this observed efficiency, one correctly edited homozygous clone was isolated for every 20 to 40 single-cell clones targeted using a single ssODN template, whereas hundreds of single cell clones were needed to isolate a biallelically edited heterozygous clone repaired using two ssODN templates35. In contrast to these results, using a single ssODN repair template containing CRISPR/Cas9-blocking PAM mutations and SpCas9-HF1 to introduce the scaleless mutation into the FGF20 locus, we found that 4 to 5 correctly edited clones were isolated for every 10 clones screened. In contrast, we were unable to isolate a clone with precise biallelic HDR using SpCas9-WT from the number of clones that we screened which suggests that many more clones will need to be picked. In our attempt to introduce the scaleless mutation into one allele ( sc/+), we were able to isolate 2 correctly edited clones containing heterozygous biallelic edits for every 10 clones screened. It must be noted that we performed single cell culturing in a growth-factors-optimised, serum-free and feeder-free culture medium11. In our protocol, chicken PGCs proliferate more rapidly (21-hr doubling time) than the PGCs cultured in high-serum chicken PGC medium10.

A major requirement for the use of SpCas9-HF1 is that the 20-nt gRNA sequence must be perfectly complementary to the genomic target to achieve high on-target editing efficiency45. When using the U6 promoter to drive sgRNA expression, the requirement for a 5′- G base in the sgRNA sequence limits the use of SpCas9-HF1 in targets that do not have a 5′G, and adding an extra G significantly reduces on on-target efficiency45,72. However, it has been shown that using alternative promoters such as the U3 promoter to express sgRNA, expressing sgRNAs from synthetic tRNA-sgRNA constructs or using hammerhead ribozyme-linked sgRNAs leads to similar levels of on-target efficiencies SpCas9-WT72,73.

In targeting the FGF20 gene with SpCas9-HF1, we found that the scaleless 535A > T nucleotide change located 6 bp downstream of the PAM and 12 bp away from the cut site of the gRNA was incorporated biallelically at a rate of >90% in isolated HDR clones containing PAM mutations. In comparison to our results, a 12 bp cut-to-edit distance was shown to result in <20% biallelic incorporation of the edit in biallelic HDR clones in human IPS cells35. Surprisingly, we also observed that all INDEL clones targeted using SpCas9-WT and Sca-ssODN did not contain the 535A > T substitution or PAM mutation suggesting that these clones never underwent HDR editing event. The only CRISPR/Cas9-blocking mutation in the Sca-ssODN template is a single nucleotide substitution in the PAM (AGG → AGA) which may not be sufficient to block re-cutting of the repaired site by Cas9. It has been shown in human cells that NGA PAMs may have up to 40% activity in some loci74,75. Interestingly, we also observed that some clones contained the 535A > T substitution on only one allele while the PAM mutation was present on the two alleles suggestive of partial or incomplete HDR and has been reported by others. This may be indicative of a cut-to-distance dependence mechanism in the incorporation of single nucleotide edits in chicken PGCs as previously reported in human cells and mouse zygotes27,35. Furthermore, we observed that Silent-ssODN was used more frequently for allelic repair when it was mixed with Sca-ssODN whereas Silent2-ssODN and Sca-ssODN were used at almost equal frequency. The absence of the distal mutation 12 bp away from the cut site in Silent-ssODN may have favoured this donor and has been previously observed in human cells35. Also, we did not see any evidence of template switching for allelic repair between Sca-ssODN and Silent-ssODN which has been reported to occur in human cells41. While we used an asymmetric ssODN donor to introduce scaleless 535A > T nucleotide change into FGF20 (Fig. 5A) based on the reported ability of this template design to increase HDR66, we are unable to tell from our results if asymmetric repair templates are more efficient than symmetric templates in enhancing HDR in chicken PGCs and therefore may require further investigation. We also tested the use of SCR7 and L755507 to increase HDR in PGCs but we did not observe any improvement or toxicity. SCR7 and L755507 are small molecules reported to increase CRISPR-mediated HDR by inhibiting NHEJ in some cell types34,53. Concentration of these inhibitors may need to be optimised for PGCs. Use of these inhibitors and other reported HDR enhancers such as RS-176 merit further investigation in PGCs.

Why we do observe such high HDR rates in avian PGCs using SpCas9-HF1? It is possible that many PGCs targeted with SpCas9-WT do not survive due to the induction of another round of cleavage of the HDR-edited site. Germ cells from many vertebrate species have been shown to undergo programmed cell death when exposed to reagents causing DSBs as a mechanism to protect the integrity of the germline genome77,78,79,80,81. In our experiments, we used plasmid delivery of SpCas9-WT which has been shown to have some toxicity in human embryonic stem cells compared to ribonucleoprotein (RNP) delivery82. In human pluripotent stem cells, it has been reported that the induction of a single DSB by SpCas9-WT is toxic even in the absence of the induction of multiple DSBs or off-target mutagenesis83. This toxicity is P53-dependent and the induction of P53 by Cas9 leads to apoptosis or cell cycle arrest in the G1 phase where NHEJ is predominant thereby reducing the efficiency CRISPR/Cas9 precision genome editing83,84. Depending on the gRNA and loci, the sustained expression of SpCas9-WT from a plasmid increases the potential for re-cleaving of HDR-edited chromosomal targets as well as off-target mutagenesis. Since the SpCas9-WT nuclease spends up to 6 hrs tightly bound to the cut ends of the DNA duplex66, the long residence time coupled with the high cleavage activity of SpCas9-WT probably increases the severity of genotoxic insult by preventing DNA repair, which may result in a stalled replication fork leading to cell cycle arrest or apoptosis and a decrease in overall HDR events. Indeed, inhibition of P53, a pro-apoptotic protein that is activated by DNA damage85, has been shown to increase the rate of HDR in human cells by preventing DNA damage response that results in apoptosis and allowing the cell cycle to progress83,84.

Conclusion

Our results demonstrate possible rapid introgression of specific haplotypes into primordial germ cells. These genomic tools will allow the validation of SNP and other chromosomal changes in poultry.

Materials and Methods

CRISPR Plasmids

PX459 V2.0 vector33 was used for expression of SpCas9-WT and sgRNA. The equivalent expression cassette (HF-PX459 V2.0) for SpCas9-HF1 and sgRNA was generated by transferring the domain containing the point mutations of SpCas9-HF1 into the coding sequence of SpCas9-WT in PX459 V2.0. A 1.4 kb region of the coding sequence of SpCas9-WT in PX459 V2.0 was excised using ApaI and PmlI restriction digest and replaced with a homologous 1.4 kb overlapping PCR fragment (Left primer, 5′-TTCCGCATCCCCTACTACGTGGGCCCCCTGGCCCGAGGGAACTCTC-3′ and right primer 5′-TCCGGGAGTCCAGGATCTGTGCCACATGCTTTGTGATGGCGCGGGTTTC-3′) from the coding sequence of SpCas9-HF1 in VP12 vector45 using NEBuilder® HiFi DNA Assembly (New England Biolabs). PX459 V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988) while VP12 was a gift from Keith Joung (Addgene plasmid # 72247).

gRNA design and CRISPR/Cas9 vector construction

gRNA sequences were selected using CHOPCHOP gRNA design web tool ( http://chopchop.cbu.uib.no/)86,87 and MIT gRNA design web tool ( http://crispr.mit.edu/) except where described otherwise. gRNA oligonucleotides were synthesized by Invitrogen and inserted into PX459 V2.0 and HF-PX459 V2.0 vectors using methods previously described in33. gRNA sequences are listed in Supplementary Table S1.

ssODN donors

ssODN donors were Ultramer® DNA Oligonucleotides synthesized by Integrated DNA Technologies (IDT). Donors were used at a concentration of 10 µM for transfections. See Supplementary Table S2 for sequences of ssODN donors.

Animal usage

Fertile eggs were obtained from a flock of commercial Hyline layer hens maintained at the Roslin Institute. The GFP+/+ germ cells used in the experiments shown in Figs 3 and 4 were obtained by crossing the Roslin Green (ubiquitous GFP) line of transgenic chicken88 to produce homozygous fertile eggs for PGC derivations. Commercial and transgenic chicken lines were maintained and bred under UK Home Office License. All experiments were performed in accordance with relevant UK Home Office guidelines and regulations. The experimental protocol and studies were reviewed by the Roslin Institute Animal Welfare and Ethical Review Board (AWERB) Committee.

PGC culture

PGC lines were derived and cultured in FAOT medium as described in Whyte et al.11. Briefly, fertile eggs from Hyline layer lines or the Roslin Green line of transgenic chickens88 were incubated for 2.5 days and then 1 μl of embryonic blood was taken from the vasculature of stage 16 HH chick embryos89 and placed into FAOT medium. FAOT medium contains 1 × B-27 supplement (Thermo Fisher Scientific), 2.0 mM GlutaMax (Thermo Fisher Scientific), 1 × non-essential amino acids (Thermo Fisher Scientific), 1 × EmbryoMax nucleosides (Merck Millipore), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific), 0.2% ovalbumin (Sigma), 1.2 mM pyruvate (Thermo Fisher Scientific), 0.15 mM CaCl ₂, 0.01% sodium heparin (Sigma), 4 ng/ml FGF2 (R&D Systems), 25 ng/ml activin A (Peprotech) and 5 µg/ml ovotransferrin (Sigma) in Avian Knockout DMEM (osmolality: 250 mOsmol/kg, 12.0 mM glucose, calcium chloride free; Thermo Fisher Scientific, a custom modification of Knockout DMEM). PGC lines were expanded to 2.4 × 10 ⁵ cells in 5 weeks before use in targeting experiments. GFP-PGCs were derived from transgenic chickens created by lentiviral methods88 and maintained at the National Avian Research Facility, Midlothian, UK.

PGC transfection

200,000 PGCs were transfected with 1.5 µg of CRISPR vector and 0.4 µg of ssODN donor using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). Cells were washed in Optimem I (Thermo Fisher Scientific) and incubated in suspension in Optimem I mixed with DNA and transfection reagent for 6 h. PGCs were then centrifuged and resuspended in FAOT medium. PGCs were treated with 0.6 µg/ml puromycin 24 h post-transfection for 48 h to enrich for transfected cells and then expanded in culture for two weeks to eliminate transient CRISPR expression and then used to isolate genomic DNA to measure targeting efficiencies or isolate single cells. To test NHEJ inhibitors, PGCs were treated with SCR7 (Sigma-Aldrich) at a concentration of 1 mM or L755507 (Sigma-Aldrich) at a concentration of 5 µM for 24 h after transfection followed by puromycin treatment for 48 h.

Isolation of single-cell clonal populations

PGCs were seeded manually or sorted using a FACSAria III (BD Biosciences) into 96-well plates at 1 cell per well in 110 µL FAOT medium and cultured for 2 weeks. Once cell density reached 30-50%, PGCs were transferred into a 48-well plate and subsequently into a 24-well plate for further expansion to isolate genomic DNA for analysis of mutation genotype.

Genomic DNA isolation and PCR amplification of target region

Genomic DNA was extracted from PGCs using QIAMP Micro Kit (Qiagen) according to the manufacturer's instruction. Specific primers for PCR amplification of sgRNA target sites were designed using primer3 software ( http://primer3.ut.ee/)90,91. Primers were designed to anneal outside the homology arms of HDR templates. List of primer sequences are listed in Supplementary Table S3. All PCR amplifications were performed using 100 ng of genomic DNA and Q5® Hot Start High-Fidelity DNA polymerase (NEB) or Phusion® High-Fidelity DNA polymerase (NEB) according to the manufacturer's protocol. Primer annealing temperatures were calculated using the online NEB Tm calculator ( https://tmcalculator.neb.com).

Analysis of gRNA targeting efficiency by T7 endonuclease I mismatch assay

T7 endonuclease I assay was performed by treating 200 ng of PCR DNA with 10 units of T7 endonuclease I in NEBuffer™ 2 buffer and incubated according to the manufacturer's instruction (NEB). The digestion products were resolved on 1% agarose gel containing Gelred® nucleic acid gel stain (Biotium) and ImageJ ( https://imagej.net) was used to quantitate band intensities. The targeting efficiency was calculated using the equation 100 × (1 − (1 − fraction cleaved)1/2)92.

HDR quantification by EcoRI restriction digestion

Restriction site sequences were incorporated into the target site to quantify HDR. All restriction enzyme digestion reactions were performed by treating 200 ng of PCR DNA with 10 units of restriction enzyme ( EcoRI-HF (NEB #R3101S), BamHI-HF (NEB #R3136S), AfeI (NEB #R0652S), AatII (NEB #R0117S)) in CutSmart buffer (NEB) incubated at 37 °C for 1 h. The digestion products were resolved on 1% agarose gel containing Gelred® nucleic acid gel stain (Biotium). ImageJ software was used to quantitate band intensities. The percentage of HDR was calculated as described in51 using the equation (b + c/a + b + c) × 100, where 'a' is the band intensity of uncleaved DNA substrate and 'b' and 'c' are the cleavage products.

HDR and INDEL quantification by flow cytometry

Live cells were gated and then GFP and BFP fluorescence was detected using a Fortessa X20 (BD Biosciences). Cytometry data was analysed using FlowJo® V7.0 (FlowJo, LLC).

DNA sequencing and bioinformatic analysis

PCR products were directly sequenced to analyse mutation genotypes of single cell clones using PCR primers or PCR products were cloned into pGEM-T Easy vector (Promega #A137A) and sequenced using T7 promoter forward primer by Sanger sequencing. Sequencing data was analysed and viewed using SeqMan Pro 13 (Lasergene 13, DNASTAR) and FinchTV 1.4.0 (Geospiza, Inc.). PCR Sanger sequencing traces were analysed with the TIDE analysis web tool ( https://tide.deskgen.com/) to detect INDELs in a population.

Data Availability

Plasmid HF-PX459 V2.0 will be available from Addgene. Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank Peter Hohenstein, Derya Ozdemir and Maeve Ballantyne for critique of the experiments and manuscript. This work was supported by Institute Strategic Grant Funding from the BBSRC (BB/P013732/1 and BB/P013759/1). AI-A was funded by a Principal's Career Development PHD Scholarship from the University of Edinburgh and PhD funding from Cobb-Vantress, Inc.

Author information

Affiliations

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK

Alewo Idoko-Akoh

, Lorna Taylor

, Helen M. Sang

& Michael J. McGrew

Contributions

A.I.-A. carried out most of the experiments. L.T. contributed to experiments. A.I.-A., H.M.S., M.J.M. planned the experiments and analysed the data. A.I.-A. and M.J.M. wrote the paper and prepared the figures.

Competing Interests

The authors declare no competing interests.

Corresponding author

Correspondence to Michael J. McGrew.

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DOI
https://doi.org/10.1038/s41598-018-33244-x

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High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells

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5 concerns from Hofstra University (babb)

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5 concerns from Hofstra University (babb)

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Through recent devolopments and discoveries in the field of biotechnology, ethics come into play. This university discusses some of the major concerns about the future of biotechnology.

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Online Master's in Health Law and Policy

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The progress of science in the 20th and 21st centuries exploded, creating technologies that reshaped society and extended lives. The pace of change was so great that it took society years to start asking questions about the ethics or morality of new developments. Now, humanity is at a major crossroads, where further investment in biotechnologies could change the way humans live and reproduce. However, the advancements may come at too steep a price.

In 2016, biotechnology engineers and researchers must address five core concerns that are the foundations of bioethics.

Use of Genetics

The Human Genome Project generated billions of dollars in funding in the 20th century, with the goal of creating a single depository of DNA base pairs and possible human genomes. On April 14, 2003, the project reached completion, and the genetic engineering field grew quickly.

Uncovering the human genome was a tremendous step forward for science, but the discovery created a series of new concerns.

Researchers have been able to use the information from the Human Genome Project to develop the new science of pharmacogentics, the study of how an individual's genes affect his or her body's ability to metabolize or react to a drug. The study of pharmacogentics aims to eliminate adverse drug events (ADE) and side effects by tailoring drugs specific to a patient's genome. While the new personalized medications can eliminate side effects, the use of genetic information to create medicine contributes to the rising cost of drugs, and shifts attention away from designing affordable drugs available for mass production.

The creation of designer babies is another critical bioethical issue for geneticists. Designer babies are children enhanced through gene manipulation to meet certain mental, physical, and emotional demands of parents. The technology does not yet exist to manipulate the entire genome of a fetus, but research continues along this path.

Stem Cells

Stem cells are the core of much biotechnical research. Because the stem cell lines can grow into any human tissue, they are invaluable for research into medicines and medical treatments, without putting people at risk. Eventually, scientists believe that stem cells may be used to grow new organs and reverse paralysis.

Almost all stem cells are the result of embryos fertilized in vitro and placed in frozen storage. Couples choose to use the embryos for implantation, though only a fraction of the embryos are ever implanted. As the cost of storage grows, couples must decide to destroy the embryo or donate them.

The use of stem cells, especially in the United States, is one of the most controversial issues in biotechnology. Supporters argue the embryos used to generate stem cell lines were going to be destroyed, and using them for research has the capacity to save untold numbers of lives. Opponents believe creating new lines from embryonic stem cells is akin to abortion, and the destruction of any embryo for research purposes is an ethical violation. Both sides have come to a partial agreement on the use of adult stem cells over embryonic cells, but the adult cells offer far fewer options and less promising research.

Human Testing

As medical and biotechnology grow, so do the pressures for companies to bring new products to the market. The FDA's new drug pipeline is clogged with thousands of drugs making their way toward approval, though only about 12 percent will ever see mass production. The approval process can take up to 10 years, and the financial stress of seeking FDA approval is too much for many companies.

One of the biggest problems with the FDA approval process is the amount of time that drugs must spend in human clinical trials. Estimates show that a new drug can spend as long as seven years in human trials. Even when the drug completes the trial, it may not receive FDA approval.

Pharmaceutical companies are calling on the FDA to speed up the clinical trial portion of the approval process, greatly reducing the time spent in the drug pipeline and increasing the profits to be made. Reducing the approval time is a double-edged sword. Medications that work will begin helping patients much sooner, but rushing through the process removes safeguards put in place to keep the public safe.

GMO Food

A growing global population, concerns about diminished drinking water reserves, and a need to keep food affordable all prompted biotechnology engineers to expand the availability of genetically modified organisms (GMOs) in the food supply. The GMOs are cheaper to produce and heartier than unmodified food, allowing farmers to meet global food needs and maintain profits.

The backlash against GMOs has been strong, and grows every day. Opponents of GMOs claim the organisms actually put the entire food supply at risk through the homogenization of plant life and the death of biodiversity. They also argue that insects and plant-destroying bacteria or diseases will continue to evolve with the GMOs, resulting in superpests and superdiseases that are untreatable by modern methods. Finally, doctors argue that GMOs include antibiotics that make their way into the human body. Overconsumption of antibiotics is harmful, because those drugs lose their ability to fight off disease.

Costs

Developments in new drugs, instruments that improve the quality of life, and extend life expectancy to new highs are all lofty goals, but they come at a significant price. The cost of health care in the United States continues to grow, largely because of innovative medical treatments and drug options. Every year more families go bankrupt trying to pay for treatments made available through biotechnology, and there seems to be little sign of lower prices in the future.

The newest designer drugs will cost patients tens of thousands, if not hundreds of thousands of dollars a year. Biotechnology engineers and companies must find a way to address cost issues and make new advancements more affordable for the average consumer; otherwise, biotechnology will create a two-tiered society: those who can afford the medical treatments needed to live, and those who cannot.

Legal Implications of these Issues

Ethical concerns of biotechnology engineering have a broad impact, influencing a number of differing careers and industries. For example, moving forward it will be essential for entrepreneurs and biotechnology companies understand the ethical and legal implications within this field so that they make well-educated decisions while still remaining innovative. The health and pharmaceutical industries are also influenced by these concerns, particularly in regards to GMO foods and human testing. Individuals and companies within these industries will greatly benefit from understanding the potential legal implications of their decisions as one wrong decision could result in a series of expensive lawsuits.

Ethics in biotechnology will always be a hot-button issue, and one the industry must take the time to discuss. Failure to do so may result in the defunding of critical projects and social backlash against important research.

To learn to speak the language of health law and distinguish your role as a leader with specialized health care compliance expertise, look to pursue a Hofstra Law online master's in health law and policy.

Sources
U.S. National Library of Medicine - Personalizing medicine with clinical pharmacogenetics EuroStemCell Physicians Committee National Library of Medicine - Genetically Modified Foods and Social Concerns The Wall Street Journal

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5 Ethical Concerns of Biotechnology Engineering

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The progress of science in the 20th and 21st centuries exploded, creating technologies that reshaped society and extended lives.

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In recent years, we've seen the development of technology that could possibly result in the concept of "designer babies" becoming reality. IVF, used commonly to help infertile couples, is the process of pre-selecting embryos before implantation. With PDG, scientists can also talk embryos less predisposed to diseases like sickle-cell anemia or cystic fibrosis before implanting in the mother

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Ethics of Designer Babies | Embryo Project Encyclopedia

# data[].attributes.attachment_link.description
A designer baby is a baby genetically engineered in vitro for specially selected traits, which can vary from lowered disease-risk to gender selection. Before the advent of genetic engineering and in vitro fertilization (IVF), designer babies were primarily a science fiction concept. However, the rapid advancement of technology before and after the turn of the twenty-first century makes designer babies an increasingly real possibility. As a result, designer babies have become an important topic in bioethical debates, and in 2004 the term “designer baby” even became an official entry in the Oxford English Dictionary. Designer babies represent an area within embryology that has not yet become a practical reality, but nonetheless draws out ethical concerns about whether or not it will become necessary to implement limitations regarding designer babies in the future.