by Matthew Cobb
Despite these very slim risks, researchers around the world were aghast at the news of the recreation of the Spanish Flu virus.39 Lord May, a former president of the Royal Society, said:
The work they are doing is absolutely crazy. The whole thing is exceedingly dangerous. Yes, there is a danger, but it’s not arising from the viruses out there in the animals, it’s arising from the labs of grossly ambitious people.
Perhaps the strongest reaction was from Simon Wain-Hobson, a virologist at the Institut Pasteur:
It’s madness, folly. It shows profound lack of respect for the collective decision-making process we’ve always shown in fighting infections. If society, the intelligent layperson, understood what was going on, they would say ‘What the F are you doing?’
This was precisely the kind of response that Berg and his colleagues feared would become widespread with the development of the new technology, and which led them to propose first the moratorium and then the adoption of stringent biosecurity measures. In response to such concerns, in October 2014 the US government introduced a temporary moratorium on funding experiments that would increase the pathogenicity of viruses. This in turn met with criticism from researchers and pharmaceutical companies who argued that our ability to respond to future pandemics might be damaged by this policy.40 Whatever the eventual outcome of this debate, the way in which this issue has been handled is in striking contrast to the self-regulation embodied by the Asilomar conference.
*
Berg’s 1972 paper on genetic engineering in E. coli raised the possibility of altering humans suffering from genetic diseases, by introducing a correct copy of a faulty gene, in a process known as gene therapy (the term was coined before Berg’s paper appeared).41 These procedures are generally directed at the affected tissues, not the germ line (eggs and sperm), so they do not alter the genes that are passed on to the next generation – several European countries have banned germline gene therapy because of uncertainty about its long-term consequences. Gene therapy was first used in 1990, and interest grew after the launch of the Human Genome Project, although renewed doubts about the safety and effectiveness of the procedure surfaced in 1999 after the death of 18-year-old Jessie Gelsinger, a patient who had received treatment for liver disease. In recent years there has been a resurgence of interest in the technique, with hundreds of clinical trials of a range of therapies, including treatments of various forms of leukaemia and retinal disease and of Parkinson’s disease, many of which have been successful. In 2012, the European Union licensed gene therapy as a treatment for a rare defect in fat metabolism.42 Although the pipeline from concept to therapy is long, complex and expensive, the future looks promising. That is certainly the view of venture capitalists, who have begun pouring hundreds of millions of dollars into the field.43
One technique that is being widely touted as a game-changer for both science and medicine is a method for directly editing the genetic code, generally known as CRISPR. The technique takes its name from the full title of the enzyme that does the work, which goes by the mouthful of ‘Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated RNA-guided endonuclease Cas9’. There are several similar enzymes found in bacteria, where they serve the function of a defence molecule, attacking and chopping up bits of invading viruses. The bacterial genome contains short palindromic repeats of twenty-four to forty-eight base pairs, which are separated by other sequences of DNA of about the same length, called spacers. The enzyme is activated by RNA transcribed from stretches of spacer DNA, which correspond to the genetic code of an invading virus that the bacterial strain has encountered in the past and which the bacteria have incorporated into their genome as a kind of memory. When a virus enters the bacterial cell and tries to hijack the bacterium’s machinery to reproduce itself, Cas9 (or a similar enzyme) is activated and attacks the virus, snipping out the bit of DNA that it recognises, thereby disabling the invader.
In 2012, Emmanuelle Charpentier and Jennifer Doudna, then based at Umeå University in Sweden and the Howard Hughes Medical Institute at Berkeley, announced that they had found out how to harness this system to change any DNA sequence.44 Within a year, CRISPR was being used to genetically manipulate DNA from a wide range of organisms, including humans.45 The principle is straightforward: the Cas9 enzyme is introduced into a cell along with a piece of synthetic RNA containing CRISPR sequences interspersed with a sequence from a gene that you are interested in rather than a bit of viral DNA. The Cas9 enzyme looks for that sequence, finds it in the genomic DNA of your organism, and snips it out. The gene of interest has either been disabled, or, if you combine CRISPR with other techniques, altered in some way. This approach is called directed mutation – targeting a particular gene in a predetermined way – and will apparently be available in virtually any organism; it is even possible to correct mistakes in the DNA sequence, such as occur in genetic diseases.46 Although the technique is in its early days, it is clearly going to revolutionise scientific discovery and may lead to the development of new gene therapies.
CRISPR looks like it will be far more effective and flexible than the previous tool of choice, RNAi (RNA interference). RNAi is based on a naturally-occurring mechanism of gene regulation that is of fundamental importance in our cells, in which short strands of RNA that complement the mRNA from a particular gene, together with a complex of proteins, block the activity of the gene by binding to its mRNA. On hearing of the CRISPR breakthrough, Craig Mello, who with Andrew Fire won the 2006 Nobel Prize in Physiology or Medicine for the discovery of RNAi, described his reaction:
CRISPR is absolutely huge. It’s incredibly powerful and it has many applications, from agriculture to potential gene therapy in humans … It’s one of those things that you have to see to believe. I read the scientific papers like everyone else but when I saw it working in my own lab, my jaw dropped. A total novice in my lab got it to work.47
If patent issues can be overcome – the Broad Institute, jointly run by MIT and Harvard, has successfully obtained a patent on CRISPR, and the two main inventors of the technique, Charpentier and Doudna, have also filed patents – then this technique could transform biology and medicine.48 Already, Doudna has extended the power of CRISPR to be able to alter RNA, thereby enabling finely tuned detection and manipulation of mRNA.49 Whatever happens next, I would bet that Charpentier and Doudna will eventually receive that telephone call from Stockholm.
A glimpse of the radical implications of CRISPR is given by the suggestion from a group of Harvard researchers that CRISPR could be used to potentially ‘prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species’.50 None of the researchers were ecologists, but they sounded the alarm about potential side-effects, and simultaneously published a call for discussion about how to regulate the new technology, coming up with criteria that should be adhered to before the implementation of any such programme, and also identifying regulatory gaps that need to be filled by legislators around the globe.51
In January 2015, the same group of Harvard researchers came up with an ingenious technofix for ensuring that GMOs with potentially problematic modifications do not cause havoc in the environment – a group from Yale simultaneously published a similar report. Both groups used a ‘genetically recoded organism’ – a special strain of E. coli in which certain codons had been manipulated to code for synthetic amino acids that are not available in the environment. These synthetic amino acids are essential to the functioning of key proteins in these organisms, which are therefore effectively restricted to living in artificial conditions. Were the bacteria to escape, they would die. Furthermore, the authors claim that the alternative genetic code used by these organisms effectively prevents horizontal gene flow. This would seem to open the road to creating potentially hazardous GMOs in the knowledge that they would be contained by their engineered physiological requirements. However, a great deal of further work will b
e needed before this approach can be applied in the real world, and I suspect few scientists – or readers – would want to rely solely on this technique to ensure biosecurity.52
These responsible approaches to the potential impact of a new technique of unprecedented power are a direct descendant of the Asilomar conference on recombinant DNA that so successfully guided science as it was catapulted into the new world of genetic manipulation. In 2008, Paul Berg reflected on the impact of the Asilomar conference:
In the 33 years since Asilomar, researchers around the world have carried out countless experiments with recombinant DNA without reported incident. Many of these experiments were inconceivable in 1975, yet as far as we know, none has been a hazard to public health. Moreover, the fear among scientists that artificially moving DNA among species would have profound effects on natural processes has substantially disappeared with the discovery that such exchanges occur in nature. … That said, there is a lesson in Asilomar for all of science: the best way to respond to concerns created by emerging knowledge or early-stage technologies is for scientists from publicly-funded institutions to find common cause with the wider public about the best way to regulate – as early as possible. Once scientists from corporations begin to dominate the research enterprise, it will simply be too late.53
Faced with a future potentially populated by CRISPR-modulated DNA-based organisms and full of bizarre synthetic life-forms that use XNA and unnatural base pairs and can record what is happening to them in their genetic material, Berg’s view, from a man who has looked at the question from both sides, is a salutary reminder for us all. His approach was to recognise the potential dangers and to find ways of countering them in conjunction with the public and regulators. The implication is that science is too important to be left to the corporations – or to the scientists.
* I’m afraid it was me.
–FIFTEEN–
ORIGINS AND MEANINGS
In May 1953, a week before Watson and Crick’s second Nature paper introduced the world to the concept of genetic information, an article appeared in Science, signed by a 23-year-old PhD student, Stanley Miller.1 Together with his supervisor, Harold Urey, Miller had attempted to discover how life might have begun on Earth. Using two connected flasks, they replicated the conditions of about 3.5 billion years ago: one flask represented the primitive ocean (sea water), the other represented Earth’s early atmosphere, and contained hydrogen, ammonia and methane (oxygen appeared in large quantities much later, and reached modern levels only about 600 million years ago). Pulses of electricity were periodically sent through the apparatus to mimic the effect of lightning. To Miller’s surprise, within a few days he could detect amino acids, in particular glycine. A simple chemical process, with no direct human guidance, had produced the components of a protein. Glycine has since been detected on a comet, showing that amino acids exist elsewhere in the Universe and could have been brought to Earth by comets shortly after the formation of the planet.2
Although the Miller–Urey experiment shows that amino acids can be formed relatively simply, it does not shed light on how life arose – we are more than just bags of amino acids. There are several scenarios for the origin of life – we do not know which is correct, and it is possible that we will never know. Here I will describe one hypothesis that is being explored by Nick Lane at University College London and Bill Martin at the Heinrich-Heine-Universität in Düsseldorf.3 According to this view, the first replicating molecules appeared perhaps 4 billion years ago in the microscopic pores of rock around a deep-ocean hydrothermal vent.* Experimental evidence shows that such pores can act as a cell, containing and constraining molecular interactions, including the accumulation of nucleotides, and also allowing compounds to be exchanged with the outside world.4
Today every cell on the planet uses electrochemical gradients to move energy around and power its activities – known as proton gradients, they are also found around hydrothermal vents, where alkaline water bubbling up from under the sea bed meets acidic sea water. According to Lane and Martin, early life, which would just have consisted of a small number of types of replicating molecule, could have used these proton gradients to gain energy. Deep in the sea, and encased in rock, these molecules would also have been protected from the destructive effects of the powerful ultraviolet radiation that bombarded the surface of the planet at that time.5
Other scenarios are available. In his 1981 book Life Itself, Francis Crick put forward a theory he developed with Leslie Orgel, in which he argued that life was the result of what they called directed panspermia. Their surprising suggestion was that life on Earth originated with microorganisms that ‘travelled in the head of an unmanned spaceship sent to earth by a higher civilisation which had developed elsewhere some billions of years ago.’6 Aside from the distinct lack of proof, this does not explain the origin of life at all – it simply puts the problem back a long time ago in a galaxy far, far away.7 It is possible that life originated elsewhere in the Universe and came to Earth on a meteorite or a comet. However, that hypothesis does not seem to be necessary – we seem to be within touching distance of understanding the chemical dynamics that created life spontaneously.
Proteins and DNA, which are so important to life today, have not always been present. The RNA machinery that exists in every cell of every organism on the planet, and the ability of RNA molecules to act as enzymes, catalysing biochemical reactions without the involvement of proteins, all indicate that another form of life existed before DNA-based life-forms: the RNA world.8 Exactly what the first replicating molecules were, and how they made the transition from merely replicating to also interacting with the world and therefore truly becoming alive, we do not know – they may have been RNA molecules, or simpler compounds, such as peptides.9 One essential feature of those early replicating systems would have been that they were able to speed up the chemical reactions that define life. The ability of molecules like RNA to act as enzymes and to catalyse reactions was discovered in the early 1980s by Sidney Altman and Thomas Cech, who won the 1989 Nobel Prize in Chemistry for their work. If left to their own devices, the kind of reactions that take place in our cells would need billions of years to occur spontaneously; in the presence of RNA they take a fraction of a second.10
At some point, perhaps after a period of evolution and competition between various biochemical types of life, the RNA world came into being.11 There are no direct traces of this world, so our views are based on strong suppositions rather than physical evidence. This was a very different kind of life. In the RNA world, RNA molecules were the basis both for reproduction and for biochemical interaction. In a world without DNA or proteins, the genetic information contained in an RNA molecule coded simply for that piece of RNA. There was therefore no code, in terms of the genetic material containing a representation of another molecule – the earliest RNA genes coded themselves and that was it. Reproduction involved the copying of RNA molecules that acted as enzymes to direct chemical reactions. These RNA molecules provided the raw material for natural selection to begin its long work of sifting between variants, eventually leading to the DNA-based life that now covers the planet.
The idea of the RNA world seems to have first been put forward by Oswald Avery’s colleague, Rollin Hotchkiss, at a symposium organised by the New York Academy of Sciences in 1957. Struck by the fact that some viruses use RNA and others use DNA, Hotchkiss suggested that
as a genetic determinant, RNA was replaced during biochemical evolution by the more molecularly and metabolically stable DNA. Cell lines have preserved the RNA entities which, evolutionwise, were primary to DNA and may have allowed them to store their information in DNA and thereby become subservient to it metabolically.12
For many years it was difficult to see how RNA could have appeared spontaneously, because the biosynthetic pathways involved in its creation seemed to be too complex. But in 2009, John Sutherland’s group, then at the University of Manchester, showed that the RNA pyrimidines (U and C n
ucleotides) could appear through a relatively simple series of reactions, using as their starting point the kind of chemicals that could have been floating about in early Earth conditions.13 We are getting closer to understanding how life might have appeared spontaneously. Already, researchers have been able to create artificial systems in which pairs of short RNA enzymes can grow and evolve in a self-sustained manner, each catalysing the growth of the other.14
Although the RNA world no longer exists (but who knows what secrets lurk in the deep ocean?), we all carry its legacy within our cells. When our DNA-based life appeared, evolution did not redesign life from scratch: it used what was to hand, adapting existing RNA biochemical pathways and turning them into something new and strange. This explains why RNA is not simply a passive messenger between the two apparently fundamental components of life – DNA and proteins. It plays many roles, shuttling genetic information around the cell and shaping how it is expressed, just as it did in the RNA world. As the RNA biochemist Michael Yarus has put it: ‘Without RNA, a cell would be all archive and no action.’15
RNA is involved in almost all of the cell’s machinery for getting the genetic information out of DNA and either creating proteins or controlling the activity of genes. In its many forms, RNA performs essential functions within the cell, even if it has lost its role as the embodiment of genetic information, replaced by the semi-inert double helix of DNA. The double helix – iconic, rigid and fixed – contrasts with the many physical forms that RNA can take, enabling it to carry out such a wide range of functions, which would have been such an important feature of the RNA world.
