2621. CRISPR Gene Editing Can Cause Hundreds of Unintended Mutations

By phys.org, May 29, 2017

CRISPR-associated protein Cas9 (white) from Staphylococcus aureus based on Protein Database ID 5AXW. Credit: Thomas Splettstoesser (Wikipedia, CC BY-SA 4.0)

As CRISPR-Cas9 starts to move into clinical trials, a new study published in Nature Methods has found that the gene-editing technology can introduce hundreds of unintended mutations into the genome.

"We feel it's critical that the scientific community consider the potential hazards of all off-target mutations caused by CRISPR, including single nucleotide mutations and mutations in non-coding regions of the genome," says co-author Stephen Tsang, MD, PhD, the Laszlo T. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology at Columbia University Medical Center and in Columbia's Institute of Genomic Medicine and the Institute of Human Nutrition.

CRISPR-Cas9 editing technology—by virtue of its speed and unprecedented precision—has been a boon for scientists trying to understand the role of genes in disease. The technique has also raised hope for more powerful gene therapies that can delete or repair flawed genes, not just add new genes.

The first clinical trial to deploy CRISPR is now underway in China, and a U.S. trial is slated to start next year. But even though CRISPR can precisely target specific stretches of DNA, it sometimes hits other parts of the genome. Most studies that search for these off-target mutations use computer algorithms to identify areas most likely to be affected and then examine those areas for deletions and insertions.

"These predictive algorithms seem to do a good job when CRISPR is performed in cells or tissues in a dish, but whole genome sequencing has not been employed to look for all off-target effects in living animals," says co-author Alexander Bassuk, MD, PhD, professor of pediatrics at the University of Iowa.

In the new study, the researchers sequenced the entire genome of mice that had undergone CRISPR gene editing in the team's previous study and looked for all mutations, including those that only altered a single nucleotide.

The researchers determined that CRISPR had successfully corrected a gene that causes blindness, but Kellie Schaefer, a PhD student in the lab of Vinit Mahajan, MD, PhD, associate professor of ophthalmology at Stanford University, and co-author of the study, found that the genomes of two independent gene therapy recipients had sustained more than 1,500 single-nucleotide mutations and more than 100 larger deletions and insertions. None of these DNA mutations were predicted by computer algorithms that are widely used by researchers to look for off-target effects.

"Researchers who aren't using whole genome sequencing to find off-target effects may be missing potentially important mutations," Dr. Tsang says. "Even a single nucleotide change can have a huge impact.”

Dr. Bassuk says the researchers didn't notice anything obviously wrong with their animals. "We're still upbeat about CRISPR," says Dr. Mahajan. "We're physicians, and we know that every new therapy has some potential side effects—but we need to be aware of what they are.”

Researchers are currently working to improve the components of the CRISPR system—its gene-cutting enzyme and the RNA that guides the enzyme to the right gene—to increase the efficiency of editing.

"We hope our findings will encourage others to use whole-genome sequencing as a method to determine all the off-target effects of their CRISPR techniques and study different versions for the safest, most accurate editing," Dr. Tsang says.

The paper is titled, "Unexpected mutations after CRISPR-Cas9 editing in vivo." Additional authors are Kellie A. Schafer (Stanford University), Wen-Hsuan Wu (Columbia University Medical Center), and Diana G. Colgan (Iowa).

2309. God’s Red Pencil? CRISPR and The Three Myths of Precise Genome Editing

By Jonathan Latham, Independent Science News, April 25, 2016

For the benefit of those parts of the world where public acceptance of biotechnology is incomplete, a public relations blitz is at full tilt. It concerns an emerging set of methods for altering the DNA of living organisms. “Easy DNA Editing Will Remake the World. Buckle Up“; “We Have the Technology to Destroy All Zika Mosquitoes“; and “CRISPR: gene editing is just the beginning”. (CRISPR is short for CRISPR/cas9, which is short for Clustered Regularly-Interspaced Short Palindromic Repeats/CRISPR associated protein 9; Jinek et al., 2012. It is a combination of a guide RNA and a protein that can cut DNA.)

The hubris is alarming; but the more subtle element of the propaganda campaign is the biggest and most dangerous improbability of them all: that CRISPR and related technologies are “genome editing” (Fichtner et al., 2014). That is, they are capable of creating precise, accurate and specific alterations to DNA.

Even the “serious” media is in on it. Nature magazine in July 2015 published “Super-muscly pigs created by small genetic tweak“. Two value judgments in a seven word headline: “small” and “tweak”, neither supported by the content of the article. Still enthralled, if not wholly original, just last week the NY Times opinion section offered: “Tweaking genes to save species“.

How do I know this is a propaganda war? I heard it from the horse itself. In February I was at a UN meeting on biotechnology in Rome, Italy, where a senior representative of the Biotechnology Industry Organisation (BIO) explained to the assembled delegates the “exquisite specificity” and “precision” of genome editing.

Myth 1: Current genome editing technologies are not error prone

BIO’s exposition is belied by the evidence. If CRISPR were already precise, accurate and specific there would, for example, be no publications in prominent scientific journals titled “Improving CRISPR-Cas nuclease specificity using truncated guide RNAs“. And these would not begin by describing how ordinary CRISPR “can induce mutations at sites that differ by as many as five nucleotides from the intended target”, i.e. CRISPR may act at unknown sites in the genome where it is not wanted (Fu et al., 2014).

Thus CRISPR itself will need tweaking before it can be useful for safe commercial products, and that is the first error of the tweaking argument. So far, it is technically not possible to make a single (and only a single) genetic change to a genome using CRISPR and be sure one has done so (Fichtner et al., 2014). As Fichtner noted “in mammalian systems Cas9 causes a high degree of off-target effects”. And at least until modified versions come into use, this will limit the safety, and hopefully limit the application, of CRISPR and related biotechnologies. There is, furthermore, no guarantee that more precise versions of CRISPR are even biologically possible. Technically therefore, precision is a myth: no form of genome editing can do what is currently being claimed.

Myth 2: Precision equals control

The second key error of CRISPR boosters is to assume that, even if we had complete precision, this would allow control over the consequences for the resulting organism.

Suppose, as a non-Chinese speaker, I were to precisely remove from a Chinese text one character, one line, or one page. I would have one hundred percent precision, but zero control over the change in meaning. Precision, therefore, is only as useful as the understanding that underlies it, and surely no DNA biologist would propose we understand DNA–or else why are we studying it?

A classic example of how DNA can still reveal unexpected functions decades after discovery is the CaMV 35S promoter, a DNA sequence used in commercialised GMO plants for almost twenty years. The CaMV 35S DNA is described in every application for commercial use as a simple DNA “promoter” (an “on” switch for gene expression).

In 1999, however, the CaMV 35S “promoter” was found to encode a recombinational hotspot (Kohli et al., 1999). In 2011 it was found to produce massive quantities of small RNAs. These RNAs probably function as decoys to neutralise the plant immune system (Blevins et al., 2011). One year later still, regulators found it to contain an overlapping viral gene whose functions are still being elucidated (Podevin and du Jardin 2012).  Will we ever know enough about any DNA sequence to accurately describe changing it as “editing”?

Myth 3: DNA functions are modular and changes are predictable

The third error of CRISPR advocates is to imply that changes to gene functions can be presumed to be discrete and constrained.

The concept of the precise editing of a genome leading to a precise biological outcome depends heavily on the conception that genes give rise to simple outputs. This is the genetic paradigm taught in schools. It is also the paradigm presented to the public and that even plays a large role in the thinking of molecular genetic researchers.

However, a defined, discrete or simple pathway from gene to trait probably never exists. Most gene function is mediated murkily through highly complex biochemical and other networks that depend on many conditional factors, such as the presence of other genes and their variants, on the environment, on the age of the organism, on chance, and so forth. Geneticists and molecular biologists, however, since the time of Gregor Mendel, have striven to find or create artificial experimental systems in which environmental or any other sources of variation are minimised so as not to distract from the more “important” business of genetic discovery.

But by discarding organisms or traits that do not follow their expectations, geneticists and molecular biologists have built themselves a circular argument in favour of a naive deterministic account of gene function. Their paradigm habitually downplays the enormous complexities by which information passes (in both directions) between organisms and their genomes. It has created an immense and mostly unexamined bias in the default public understanding of genes and DNA.

This is not my argument. It belongs to Richard Lewontin of Harvard University, probably the most famous geneticist of our time.

The benefits of naive genetic determinism to the architects of the genome-industrial complex are very great. Since it pretty much requires that organisms be seen as robots being operated by mini-dictators (rather than, for example, as systems with emergent properties) and those genes as having effects that are narrow and clearly defined rather than being diffuse and unpredictable, it simplifies their sales pitch and frames risk assessment as unnecessary.

The problem comes to a head, however, when this narrow conceptualisation of genetics is applied to the real world and situations that have not been, as it were, set up in advance. In the case of the “Super-muscly” pigs reported by Nature, strength is not their only feature. They must also have more skin to cover their bodies and stronger bones to carry themselves. They also, apparently, have difficulty giving birth; and if they were ever released into the wild, they would presumably have to eat more. Thus a supposedly simple genetic tweak can have wide effects on the organism throughout its lifecycle.

Nature also revealed that thirty of the thirty two pigs died prematurely and only one animal was still considered healthy at the time the study authors were interviewed. So much for precision.

The neverending story

Why is this discussion of precision important? Because for the last seventy years all chemical and biological technologies, from genetic engineering to pesticides, have been built on a myth of precision and specificity. They have all been adopted under the pretense that they would function without side effects or unexpected complications. Yet the extraordinary disasters and repercussions of DDT, leaded paint, agent orange, atrazine, C8, asbestos, chlordane, PCBs, and so on, when all is said and done, have been stories of the steady unraveling of a founding myth of precision and specificity.

Nevertheless, with the help of industry propagandists, their friends in the media, even the United Nations, we are once again being preached the gospel of precision. But no matter how you look at it, precision is a fable and should be treated as such.

The issues of CRISPR and other related new “genome editing” biotechnologies are the subject of intense activity behind the scenes. The US Department of Agriculture has just explained that it will not be regulating organisms whose genomes have been edited since it doesn’t consider them to be GMOs at all. The EU was about to call them GMOs but the US has caused them to blink, meanwhile the US is in the process of revisiting its GMO regulatory environment entirely. Will future safety regulations of GMOs be based on a schoolboy version of genetics and an interpretation of genome editing crafted in a corporate public relations department? If history is any guide it will.

References

Blevins, Todd, Rajendran Rajeswaran, Michael Aregger, Basanta K. Borah, Mikhail Schepetilnikov, Loïc Baerlocher, Laurent Farinelli, Frederick Meins Jr, Thomas Hohn and Mikhail M. Pooggin (2011) Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Research 39: 5003-5014.
Franziska Fichtner, Reynel Urrea Castellanos, and Bekir Ülker  (2014) Precision genetic modifications: a new era in molecular biology and crop improvement. Planta 239: 921-939 (doi:10.1007/s00425-014-2029-y.
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826.
Fu Y, Jeffry D. Sander, Deepak Reyon, Vincent M. Cascio, and J. Keith Joung (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 32: 279–284.
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmanuelle Charpentier (2012) A Programmable Dual-RNA – Guided DNA Endonuclease in Adaptive Bacterial Immunity Science 337: 816-821.
Podevin N and  du Jardin P (2012) Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants. GM Crops and Food 3: 1-5.

Further reading (added April 26): Are new biotechnologies GMOs?

2099. A New Era of Humanity's Dominion: Gene Editing

By Amy Harmon, The New York Times, November 26, 2015

A genetically modified salmon (on the top) and a regular one.

SIOUX CENTER, Iowa — Other than the few small luxuries afforded them, like private access to a large patch of grass, there was nothing to mark the two hornless dairy calves born last spring at a breeding facility here as early specimens in a new era of humanity’s dominion over nature.

But unlike a vast majority of their dairy brethren, these calves, both bulls, will never sprout horns. That means they will not need to undergo dehorning, routinely performed by farmers to prevent injuries and a procedure that the American Veterinary Medical Association says is “considered to be quite painful.”

Instead, when the calves were both just a single cell in a petri dish, scientists at a start-up company called Recombinetics used the headline-grabbing new tools of gene editing to swap out the smidgen of genetic code that makes dairy cattle have horns for the one that makes Angus beef cattle have none. And the tweak, copied into all of their cells through the normal machinery of DNA replication, will also be passed on to subsequent generations.

“It’s pretty cool,” said Micah Schouten, the calves’ caretaker, looking at his charges.

The uproar over the new ease and precision with which scientists can manipulate the DNA of living things has centered largely on the complicated prospect of editing human embryos. But with the federal government’s approval last week of a fast-growing salmon as the first genetically altered animal Americans can eat, a menagerie of gene-edited animals is already being raised on farms and in laboratories around the world — some designed for food, some to fight disease, some, perhaps, as pets.

Just this week, researchers reported having edited mosquitoes so that they will no longer carry the parasite that causes malaria. And the power to reshape other species, scientists and bioethicists say, raises questions that are both unique to animals and may bear on the looming prospect of fiddling with our own.

“We’re going to see a stream of edited animals coming through because it’s so easy,” said Bruce Whitelaw, a professor of animal biotechnology at the Roslin Institute at the University of Edinburgh. “It’s going to change the societal question from, ‘If we could do it, would we want it?’ to, ‘Next year we will have it; will we allow it?’ ”

Animal breeders have for centuries scoured species for desirable traits and combined them the old-fashioned way, by selective mating. But that process can take decades to achieve a particular goal, like cows that are both resistant to disease and produce a lot of milk. And until recently, genetic engineering techniques used to manipulate DNA had been so imprecise as to make them too expensive and difficult to perform in many animals.

But the new techniques, collectively called “gene editing” to reflect the relative ease of their use, have made all manner of previously impossible or impractical goals sufficiently fast and cheap for many to find worth pursuing. Using enzymes that can be directed to cut DNA at specific locations, they allow scientists to remove and replace bits of genetic code more or less on demand. “It’s like a find-replace function in the genome of these animals,” said Scott Fahrenkrug, the chief executive of Recombinetics, based in St. Paul. “It allows us to find the natural variation that exists across a species and quickly bring it under one hood.”

At Roslin, for instance, Dr. Whitelaw has changed three genes in domesticated pigs vulnerable to African swine fever, which can devastate herds, to resemble those from wild pigs that are resistant to the disease. He is now breeding them to put them to the test.

With a tool called Talens, Recombinetics says it has created gene-edited pigs that can be fattened with less food and Brazilian beef cattle that grow large muscles, yielding more meat that may also be more tender. Others are working on chickens that produce only females for egg-laying and cattle that produce only males, since females are less efficient at converting feed to muscle.

Chinese researchers have produced meatier cashmere goats that also conveniently grow longer hair for soft sweaters, miniature pigs lacking a growth gene to be sold as novelty pets and bulky beagles lacking a muscle-inhibiting gene, an edit that could make for faster dogs.

Using the most powerful of the new tools, called Crispr-Cas9, in pursuit of treatments for human disease, researchers are also altering pigs in hopes of making them grow human organs and creating “gene drives” that would ensure that the edit to make mosquitoes malaria-proof, for instance, would spread through the whole population.

An Accelerating Pace

But the rapid advent of gene-edited animals threatens to outstrip public discussion of their risks and benefits, some scientists and bioethicists have warned.

“This essay is, in essence, a plea — let’s not ignore the nonhuman part of the biosphere,” Alta Charo of the University of Wisconsin and Henry T. Greely of Stanford University cautioned in an article titled “Crispr Critters and Crispr Cracks,” to be published in The American Journal of Bioethics next month. “Not only is it much larger than the human part, but it is much more susceptible to unobserved or unfettered — but not unimportant — changes.”

The discussion of gene-edited animals in farming, in particular, will most likely be colored by the existing debate over the merits of genetically engineered food, which for decades has largely centered on corn and soybeans, altered with older technology to resist pests and tolerate herbicides. Opposition to such crops, known as genetically modified organisms, or GMOs, has prompted some retailers to decline to sell food made with them, and efforts to pass legislation to label them, even as farmers have widely embraced them and scientific organizations have said they are as safe for human health and the environment as conventional crops.

Many of the new generation of edited animals do not contain DNA from another species, a frequently cited concern among opponents of genetically engineered foods, which incorporate genes from bacteria. But some consumer advocates say it may be even more difficult to reach consensus on what, if anything, should be done to the DNA of animals.

“Animals on some level will always be more controversial,” said Greg Jaffe, director of biotechnology for the Center for Science in the Public Interest, a nonprofit consumer advocacy group. “If only because people think of them as closer to humans.”

Advocates of the technology argue that it can make farming more efficient to help feed a growing world population with less of a toll on the environment. One projection published in a leading animal breeding journal, Genetics Selection Evolution, suggests that genome-editing could significantly increase the efficiency the livestock industry is able to achieve through conventional breeding within the same time period.

Today’s chickens, for instance, produce nearly 80 percent more meat for the same amount of feed as the chickens of the 1950s; if chicken breeders had had access to genome technology over that time, said John Hickey, a quantitative geneticist and a co-author of the paper, farmers would have been able to achieve that increase and also be able to grow chickens on half the land.

Others say the technology could benefit human health. The National Science Foundation is underwriting an effort to create dairy cattle that can resist a parasite that causes sleeping sickness in sub-Saharan Africa, a blight often treated with an antimicrobial drug that ended up making its way into the meat consumed by humans.

Several projects underway to edit genetic resistance to a variety of diseases in livestock could theoretically reduce the overuse of antibiotics, which has made it harder to treat human bacterial infections. With funds from the United States Department of Agriculture, Bhanu Telugu, a University of Maryland researcher, is trying to design pigs so they can no longer serve as a reservoir for the flu virus. He argues for genome editing on behalf of animal health, too. “If we know we can eliminate the disease and we don’t, it is in my mind animal cruelty,” he said.

Fallout in the Food Chain

Still, some consumer advocates urge caution in applying techniques that are still so new to animals that will be consumed as food. Gene-editing tools are known to sometimes make changes to genes other than their intended targets, raising flags about how the changes might affect an animal’s health or the composition of milk or meat.

“You are reducing the universe of potential risks by moving into these techniques,” said Doug Gurian-Sherman, a senior scientist at the Center for Food Safety, a consumer advocacy organization that has been at the forefront of opposition to genetically engineered plants and animals. “But that is not to say we should not still proceed with great caution.”

And some animal rights advocates say gene-editing is simply a means to prop up an industry that causes animals to suffer.

“Even if they can point to good intentions, it’s just exacerbating the problem,” said David Byer, a spokesman for People for the Ethical Treatment of Animals. The organization, which has urged the dairy industry to stop the practice of dehorning cattle, does not support gene-editing as a solution.

“People should stop consuming dairy or meat or eggs, not further manipulate animals by playing with their DNA,” Mr. Byer added.

The Food and Drug Administration has not said how or whether it will regulate the gene-edited animals to come. But even with the government’s stamp of approval, biotechnology advocates know that farmers are unlikely to embrace technology if they fear consumers will reject it.

And it has not helped the popularity of genetically engineered crops that their chief benefits so far — easier control of weeds and pests for corn and soybean farmers — are not terribly compelling to the eating public.

That is one reason Recombinetics has begun to show off its hornless calves.

Dehorning, which involves burning off horn-buds to stop the flow of blood to the horn tissue, has already garnered a degree of popular concern. Videos of the burning procedure carried out on Holsteins, the black-and-white breed largely responsible for the nation’s milk supply, and circulated by animal rights groups, draw long strings of critical comments.

“We know there’s a negative public perception of dehorning, and it’s certainly not a fun chore for the farmers,” said Lindsey Worden, the executive director for genetics at the Holstein Association.

A small fraction of Holsteins are naturally hornless, and several companies, including General Mills, Dannon and Walmart, have encouraged their dairy suppliers to increase their population through conventional breeding. Farmers have made some headway, with the population of hornless Holsteins climbing to about 4 percent last year from 3 percent in 2013.

But it is slow going. That is why several dairy breeders say they are keeping tabs on Recombinetics’ two hornless calves, which have just been shipped to the University of California, Davis, to be monitored for their health. There, in a few months, their sperm will be harvested, each with edited DNA, which will be used to create a new generation of hornless cattle.

Whether they will become commonplace or remain curiosities may depend largely on how the public comes to view gene editing and its various applications.

“Sometimes you can have nice benefits for animals and farmers and society but still have controversy among consumers,” said Jamie Jonker, vice president for sustainability and scientific affairs at the National Milk Producers Federation. “I think dairy farmers are going to want to see how this is interpreted by the general public.”