Life's Greatest Secret (43 page)

The ability to manipulate DNA has recently been extended to altering the genetic code itself. Although only two base pairs occur in nature (A binds with T and C binds with G), unnatural base pairs can be used to make novel forms of DNA in test tube reactions. More than twenty-five years ago, Steven Benner’s research group extended the alphabet of the genetic code by introducing two new base pairs into DNA and RNA molecules – one pair is known as κ and Π, the other as iso-G and iso-C.
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In 2011, Benner’s group was able to amplify and sequence DNA containing the four usual bases and an unnatural base pair (Z and P), which they called GACTZP DNA.
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For decades, the manipulation of base pairs has been used in some forms of everyday antiviral medicine, such as those regularly used by cold sore sufferers. The cold sore virus is persuaded to replace the G bases in its sequence by one of several proprietary molecules that cannot be copied by the infected cell’s machinery, thereby blocking reproduction of the virus.
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In 2014, a group of researchers led by Floyd Romesberg of the Scripps Research Institute in California took a giant step towards the creation of a truly synthetic organism when they were able to make
Escherichia coli
bacteria copy a piece of DNA containing a sequence that involved an unnatural base pair (the two new bases go by the unfriendly names of d5SICS and dNaM), which had previously been created in a test tube.
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Where the
E. coli
DNA replication machinery found a d5SICS in the artificial molecule, it inserted a dNaM on the new complementary strand, and vice versa. The bacteria seemed quite happy with this new alphabet, showing no serious problems, and the classic DNA repair pathways in the cell, which normally snip out and repair errors, did not make any move against the intruding pairs of bases.

For the moment, this remains at the level of a technical breakthrough – as the title of the paper put it, the researchers had created ‘A semi-synthetic organism with an expanded genetic alphabet’. The two extra letters that were introduced into the DNA are currently mute; the expanded genetic alphabet does not yet form new words. But the authors made clear that their aim is to create a system in which unnatural base pairs will code for unnatural amino acids. The possibilities for synthetic biology – using living cells to produce new molecules – are almost endless.

It is inevitable that these astonishing developments in our ability to manipulate the essential elements of life will soon come together. Someone will eventually create a system in which XNA carries unnatural base pairs that code for unnatural amino acids that are assembled into bizarre proteins, and an utterly novel form of life, created entirely through human ingenuity, will be only a step away. It does not seem too outlandish to imagine that by the end of the century, entirely synthetic life-forms will exist, able to produce drugs, foods and novel compounds at the service of humanity, perhaps in the most inhospitable of environments. Synthetic organisms able to survive in low levels of oxygen and cold temperatures might be one way in which we could terraform Mars, should it prove to be barren, and should we consider it ethical to destroy such a pristine environment. In this respect, as others, science and technology pose questions; they do not necessarily provide the answers.

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Despite the optimism that surrounds synthetic biology and genetic engineering, ever since the first appearance of these techniques in the 1970s scientists have consistently expressed concern about the potential dangers. With the development of restriction enzymes (proteins that will snip a piece of DNA in two at a defined sequence) it became possible to create what is known as recombinant DNA – DNA from more than one organism, generally from two different species. This led scientists, including those involved in pioneering the approach, to be concerned that the introduction of new genes into organisms could have unforeseen consequences. They were particularly worried about what would happen if the organisms escaped and transferred their genes into the wild or if the new genes were inherently dangerous to humans or the ecosystem. This was not just an abstract concern: the era of genetic engineering was heralded by the introduction of
SV40,
a viral gene that can cause cancer in rodents, into the DNA of a bacteriophage virus, which was then used to transform
E. coli.
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Faced with the novelty of this technique, it was legitimate to worry that the transformed
E. coli
might end up inducing cancer in humans. The experiment, by Paul Berg, David Jackson and Robert Symons, was published in 1972. Eight years later, Berg won the Nobel Prize in Chemistry for this feat, together with Wally Gilbert and Fred Sanger, who were recognised for their work on DNA sequencing. Within a year of publication, Berg, along with other scientists, was arguing for a partial moratorium on recombinant DNA research because of the potential dangers.
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In February 1975, a conference took place at Asilomar, on the edge of Monterey Bay in California, to discuss the risks associated with the new technique and above all how to minimise the dangers. The conference, which included journalists and lawyers among the attendees, adopted a set of laboratory procedures, including strict containment facilities and biosecurity measures, which would enable research to continue safely. Many of these are still in force, but others have been abandoned as it has been realised that the dangers are far less than was originally feared.
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In my own field, the study of behaviour in
Drosophila,
the introduction of DNA from other species into the fly’s genome has become widespread in order to mark and manipulate tissues, enabling us to turn genes on and off, simply by allowing two flies to mate. The technique is perceived as entirely risk-free, and recombinant fly stocks, which may contain genes from yeast, jellyfish or bacteria, are routinely sent around the world by ordinary post and are used in ordinary laboratories, with no restrictive containment procedures.

However, genetic engineering can pose very real dangers. In June 2014, a group of US and Japanese scientists, led by Yoshihiro Kawaoka of the School of Veterinary Medicine at University of Wisconsin-Madison, attempted to recreate the Spanish Flu virus, which killed millions of people after the First World War.
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As is well known, we are in danger of another global flu pandemic, with avian flu being the most likely source because it seems also to have been the source of the Spanish Flu. Kawaoka and his colleagues took bits of avian flu virus that were similar to the Spanish Flu infectious agent and put them together in a new DNA sequence, which proved to be highly infectious, just as the Spanish Flu virus was.

They justified their study by arguing that it would help identify which are the most dangerous parts of the viral genome and would therefore increase our preparation to meet any future outbreak. Although the US National Institute of Allergy and Infectious Diseases, which funded the study, defended the research both in terms of the information it provided about the potential dangers of newly emerging flu strains and the stringent biosecurity measures that were applied, there are clear dangers. The newly created virus could escape, or it could conceivably be used by a hypothetical group of bioterrorists, although this would require them to breach the stringent security procedures around such facilities and to be highly trained microbiologists.

Despite these very slim risks, researchers around the world were aghast at the news of the recreation of the Spanish Flu virus.
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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.
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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.

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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).
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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.
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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.
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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.
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Within a year, CRISPR was being used to genetically manipulate DNA from a wide range of organisms, including humans.
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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.
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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.

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.
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Already, Doudna has extended the power of CRISPR to be able to alter RNA, thereby enabling finely tuned detection and manipulation of mRNA.
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Whatever happens next, I would bet that Charpentier and Doudna will eventually receive that telephone call from Stockholm.