Hacking the Code of Life

by Nessa Carey

  Reviewing the state of knowledge of the bacterial strains and the viruses, Mojica was able to conclude that there was a correlation between the presence of a specific spacer in a bacterium and its resistance to infection by the virus that contained the same spacer. This led him to speculate that the spacers were somehow part of an immune response that the bacteria had developed to give them protection against aggressive invaders.

  Mojica tried to get his findings published for a year and a half. It’s really important to scientists to publish in prestigious journals. This raises your profile, shows you are successful, improves your access to grant funding and also increases the chances that other researchers will read your work, learn from it and push the science forwards. But every high-profile journal that Mojica approached turned down his manuscript. Eventually, disheartened and worried that someone else would find the same connection and beat him to the publication, he published in an obscure journal in 2005.5

  It was probably a wise decision to publish, as a few other researchers were also developing an interest in these odd bacterial sequences. Like Mojica, they weren’t thinking about creating gene editing technologies. They had stumbled across the sequences while investigating new ways of monitoring germ warfare agents, or improving the commercial production of yoghurt.6 Like Mojica, these additional researchers also speculated that the repeats were somehow used by bacteria to protect themselves against infection by viruses. It also became clear that there were protein-coding genes in the same regions of the bacterial genomes as the strange repeats, although at first it wasn’t exactly clear what the proteins actually did.

  In 2007, the scientific community woke up to the importance of the bacterial sequences when a paper was published in one of the world’s leading journals, Science, which demonstrated that the repeats did indeed confer protection from viruses, and that this also required the activity of the proteins encoded by the nearby bacterial genes. Basically, if a bacterium survived an assault by a virus, it copied parts of the viral genes and inserted them into its own genome, as the 36-letter spacers in the repeat regions. This gave the bacteria resistance to any subsequent attacks by the same virus.7

  Now the pace of investigation really began to hot up. Scientists demonstrated that during a viral infection, the bacterium copied its own versions of the relevant repeats, specific to the virus which was attacking it. These copies bound to the matching region in the viral genome. Once this happened, one of the proteins that was encoded by a gene in the bacterial DNA near to the repeats attacked the viral DNA and destroyed it, bringing the viral infection to a halt.8

  Until this point, all the research had been carried out by people interested in bacteria and in how they protect themselves against viruses. But by 2008, at least some authors were starting to speculate about wider implications. The experimental data from bacteria made it clear that the repeats themselves were essential for the immunity function, and had to stay basically constant. But scientists could replace the naturally existing spacer regions with new spacers, and provided they could find a match in a viral genome, the system would still break down the viral DNA. In other words, the spacers were swappable cassettes, and this might allow scientists to destroy any matching DNA sequence they wanted.9

  The number of research labs working on this system of bacterial immunity began to increase as the novel and intriguing nature of the mechanisms at play began to capture the imagination. The fine details of how the system operated in bacteria were teased out, defining exactly which bits of the repeat regions, and which proteins, were required to make the system work perfectly.

  The blockbuster paper was published online in Science on 28 June 2012.10 It was a combined effort from the labs of Emmanuelle Charpentier and Jennifer Doudna, and drew particularly on earlier work from Charpentier that had identified another DNA sequence in bacteria that was critical for the adaptive immunity response. There were three remarkable achievements in the paper from the two women. The first was that the scientists simplified the system. In the natural situation in bacteria, the micro-organism needed to create copies of at least two different regions of its genome to target the viral DNA. Charpentier and Doudna created a hybrid version such that only one molecule, containing both regions, was required. They also showed that just one of the nearby proteins was required in order to drive the destruction of the ‘enemy’ DNA. Their third great achievement was that they were able to get the system to work in a solution, rather than in bacteria.

  This was a breathtaking development. By making the system straightforward and operational in a test tube, Charpentier and Doudna had liberated this technology. It was no longer restricted to the world of bacteria. The two women were highly attuned to the implications of their findings, speculating in the Abstract of their paper that their finding ‘highlights the potential to exploit the system for … programmable genome editing’. But to be truly useful, the system would need to work inside cells.

  Just seven months later, a paper from the lab of Feng Zhang was published in the same journal, which demonstrated that this new approach did indeed work in cells, including human ones.11 The ability to hack the code of life had truly arrived.

  How gene editing works

  This new technology that allows scientists to hack the genomes of any organism on the planet with remarkable speed, ease, precision and cheapness is actually surprisingly straightforward in its basic principles. In its original version it basically used the protocols and materials created by Charpentier and Doudna, relying on just two main foreign components.

  One of those two components is called the guide molecule. It’s made from a molecule called RNA, which is related to DNA. Like DNA, it is composed of four letters. Unlike DNA, it’s single-stranded whereas DNA is double-stranded. Where DNA forms the iconic double helix, composed of two strands of DNA letters binding to each other, RNA is a singleton. There’s only one strand and this is an important factor in its activity in gene editing.

  Let’s imagine DNA as a giant zip, where each tooth is one of the four letters of the genetic code. During gene editing, the guide RNA molecule slides along the giant zip, trying to force its way in between the teeth. Most of the time this will be impossible, but if the guide finds a region where its own sequence of letters is the same as that in the DNA, the guide molecule pushes its way into the double helix. It’s easy to use our knowledge of the genome to create a guide molecule that will bind to only one DNA sequence, for example a mutation that leads to a disease.

  The guide molecule is now in position where we want it, and the targeting phase of gene editing is complete. This relies on the second component which is a protein that can act like a pair of molecular scissors, cutting across the DNA double helix. These scissors don’t cut randomly; they don’t just flail across the genome. Instead, they only cut where the guide molecule has inserted itself into the DNA. This is because the guide molecule also contains a sequence that the scissors recognise. Only after the scissors have bound to the interloping guide molecule do they snip across the DNA. The basic process is shown in Figure 2.

  This cut damages the DNA, but all cells contain mechanisms to repair DNA very quickly. In fact, the repair mechanisms often prioritise speed over accuracy and the repair is a bit of a botch job. The two loose ends of DNA get joined together but the join isn’t quite the same as the original sequence of letters. The end-result of this is usually that the gene is no longer functional.

  This was the first iteration of what we now refer to as gene editing* and we can envisage this using our earlier analogy of the business card, which has been mistakenly printed to refer to an ‘inferior designer’. Using the first version of gene editing, extra letters would be inserted into the inappropriate word, or deleted from it. ‘Inferior’ might be altered to ‘inferantior’ or ‘inior’. Both of these are clearly nonsense and would at least stop the person reading the card from assuming that you are rubbish at furniture selection and room layouts.

  Figure 2.
The basic principle of gene editing

  The two key components are the single-stranded guide molecule and the enzyme (scissors) that can cut DNA. The guide molecule is chemically synthesised and its sequence of letters matches the gene that the researcher wants to change. When the two key components enter the nucleus of the cell, the guide molecule binds to its matching DNA sequence. The scissor enzyme cuts the DNA close to the interpolated guide sequence, and normal cellular repair mechanisms rejoin the cut ends, leaving out the fragment that matched the guide. This changes the DNA sequence. All types of gene editing are based on this principle, although numerous adaptations have been made so that increasingly precise alterations can be created, e.g. the replacement of just one letter of the DNA alphabet with another.

  Adapted from an image by Reuters; Nature; MIT

  This might seem of limited use in printing, but in genetics it’s a fantastic way to stop a gene from working. This can be remarkably useful. It allows scientists to test hypotheses about what a specific gene does in a cell or organism, and could even be useful therapeutically if a mutated gene codes for a dangerous protein.

  Of course, you have to be able to get the guide RNA and the cutting protein into the cells you want to change but this isn’t especially difficult, at least in a lab. This is often achieved by co-opting a simple virus that is very good at entering cells but doesn’t actually cause any harm to the host. Scientists package the two components required for gene editing into the virus and then infect the target cells. Once inside the cells, the virus releases its payload and the gene editing process begins.

  One of the many good things about this technique is that once a change has been engineered into the genome, the change is there for ever. Gene editing introduces permanent alterations into the DNA. It doesn’t matter if the viral Trojan horse gets broken down, or the guide RNA and protein scissors degrade – the change in DNA sequence will persist.

  In cells that don’t divide, such as neurons or heart muscle cells, the alteration to the genome will survive for as long as the cell does. In cells that do divide, the alteration will be passed on to all subsequent generations of the cells. It’s a one-hit wonder that lasts for ever.

  The earliest versions of gene editing immediately provided scientists with a hugely improved technology for inactivating genes. But researchers are never satisfied and this basic system has been hacked spectacularly by laboratories throughout the world. They have improved and extended the basic toolkit. It’s now possible to create perfect repairs, changing just one letter in the 3,000,000,000 of the human genome. In our business card analogy, we would actually be able to change ‘inferior designer’ to ‘interior designer’.

  You can go further. If you want to change just the gene inherited from Mother, and not the one inherited from Dad, that’s possible too. Maybe you don’t want to switch off a gene or change its sequence, you just want to change its levels of expression? Well, good news, you can use gene editing to do that too.

  The numbers of scientists and labs able to modify the book of life has increased exponentially since Charpentier and Doudna broke gene editing out of bacteria and into the wider world in 2012. Let’s take a look at some of the things they’ve been up to.

  Notes

  1. https://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer

  2. Adamson, G.D., Tabangin, M., Macaluso, M., Mouzon, J. de. ‘The number of babies born globally after treatment with the assisted reproductive technologies (ART)’ . Fertility and Sterility (2013); 100(3): S42.

  3. Mojica, F.J.M., Díez-Villaseñor, C., Soria, E., and Juez, G. ‘Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria’. Mol. Microbiol. (2000); 36: 244–246.

  4. For a description of Mojica’s lonely work in the early days, see Mojica F.J.M., Garrett R.A. ‘Discovery and Seminal Developments in the CRISPR Field’. In: Barrangou R., Van Der Oost J. (eds). CRISPR-Cas Systems (2013); Springer, Berlin, Heidelberg.

  5. Mojica, F.J., Díez-Villaseñor, C., García-Martínez, J. et al. J. Mol. Evol. (2005); 60: 174. https://doi.org/10.1007/s00239-004-0046-3

  6. For an interesting but rather partial review see: Lander, E.S. ‘The Heroes of CRISPR’. Cell (14 January 2016); 164(1–2): 18–28.

  7. Rodolphe Barrangou, Christophe Fremaux, Hélène Deveau, Melissa Richards, Patrick Boyaval, Sylvain Moineau, Dennis A. Romero, Philippe Horvath. ‘CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes’. Science (23 March 2007); 1709–1712.

  8. Stan J.J. Brouns, Matthijs M. Jore, Magnus Lundgren, Edze R. Westra, Rik J.H. Slijkhuis, Ambrosius P.L. Snijders, Mark J. Dickman, Kira S. Makarova, Eugene V. Koonin, John Van Der Oost. ‘Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes’. Science (15 August 2008); 960–964.

  9. Marraffini, L.A., and Sontheimer, E.J. ‘CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA’. Science (2008); 322: 1843–1845.

  10. Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmanuelle Charpentier. ‘A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity’. Science (17 August 2012): 816–821.

  11. Le Cong, F. Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano A. Marraffini, Feng Zhang. ‘Multiplex Genome Engineering Using CRISPR/Cas Systems’. Science (15 February 2013); 819–823.

  * This technology is called CRISPR-Cas9 and most versions of gene editing rely on this basic mechanism. Unless otherwise stated in the text we’ll use ‘gene editing’ as a catch-all phrase for all technologies that use this approach or variants thereof.

  3

  FEED THE WORLD

  The number of humans on our planet is increasing all the time. The world population reached 1 billion around the year 1800; in 1930 it was 3 billion; 5 billion in 1987 and today the number is in the region of 7.6 billion and rising all the time.1 Barring a meteorite strike, we will reach 8 billion in 2023, according to the predictions of the United Nations.2

  Ask most people if these increasing numbers are a problem and they will answer ‘yes’. They’re right. We are a pest species, destroying our environments and wiping out vast numbers of other organisms with whom we share this delicate globe. Ask most individuals from the economically-developed world what we need to do about this problem, and the answer is usually the same: ‘People need to stop having so many children.’

  There are two major difficulties with this response. The first is that ‘people’ usually refers to other people, typically in the less developed world. This is fairly ridiculous, as the environmental impact of children in the most economically developed countries is much higher than those from less privileged regions. A typical American has 40 times the carbon footprint of someone from Bangladesh, for example.

  The other difficulty with the ‘people need to stop having so many children’ response is that it ignores a critical fact. It’s not the number of people being born that is really the problem for our planet, it’s the number of us failing to die in a timely fashion that’s the key issue.

  Let’s imagine a couple aged 25 who decide to have two children. Two children’s a reasonable number, right, because that just replaces the parents when they die? Fast forward 25 years and our original couple are only 50, and now they are grandparents, because each of their children also decided to have kids. But they were responsible, just like their parents. Only two kids each. And 25 years on, the original couple are 75, and now they have two children, four grandchildren and eight great-grandchildren. There are now sixteen people on the planet, where once there were two.

  Birth rates are actually falling, and have been for quite some time. In 1950, the average global birth rate was 37.2 births/1,000 people each year. It’s now about half that, at 18.5 births/1,000 people each year.3,4 Death rates have shown the same trends in the same period, dropping from 18.1 deaths/1,000 people
each year in 19505 to 8.33 deaths/1,000 people each year in 2017.6

  Based on current mortality rates, life expectancy in the UK has risen to 79.2 years for men and 82.9 years for women.7 In 1951, the figures were 66.4 and 71.5 years respectively.8

  As long as the death rate is lower than the birth rate, the world’s human population will continue to grow. The rate of growth of the global population will decrease if the birth rate keeps falling, but the numbers will keep going up for the foreseeable future.

  The consequences of the ever-increasing numbers of humans on this planet are horrifying, with competition for resources intensifying all the time. One of the areas of peak concern is how to feed everyone, and also how to do this without destroying the ecosystems that we will rely on in the future.

  Although it’s often claimed that we can’t produce enough food for the world’s human population, this isn’t actually true. We certainly can’t produce enough food to feed everyone the spectacularly unhealthy western diet that rapidly becomes the norm as societies become more affluent. Average per capita consumption of meat in the industrialised world is 88kg per person per year, compared with 25kg in the less developed economies.9 Unless foraging in low-impact systems, animals inevitably require more inputs than plants to produce a given quantity of human food. At its extremes, in intensive rearing systems, as much as 7kg of grain can be required for each kilogram of beef meat produced.

  So we probably can’t support western levels of meat consumption and we certainly can’t support western levels of general over-consumption. 64% of adults in England are overweight, obese or morbidly obese.10 The figure for the US is even higher at 70.2%.11 A grotesque consequence of this is that we will almost inevitably see global death rates start to rise, and life expectancy start to fall, slowing the rate of population growth. But the overall numbers of us on this planet will continue to increase for many years.