Long-live the Revolutions!!

There’s a general view that most folk don’t know much about science and, because almost day by day, science plays a more prominent role in our lives, that’s considered to be a Bad Thing. Us scientists are therefore always being told to get off our backsides and spread the word – and I try to do my bit in Betrayed by Nature, in Secret of Life (a new book shortly to be published) and in these follow-up blogs.

We may be making some progress – and, I have to admit, television has probably done more than me – though I am available (t.v. & movie head honchos please note). As one piece of evidence you could cite the way ‘DNA’ has become part of the universal lexicon, albeit often nonsensically. As evidence I call Sony Corp. Chief Executive Kazuo Hirai, as reported in The Wall Street Journal: “I’ve said this from day one. Some things at Sony are literally written into our DNA …”

Well, of course, that’s gibberish Kazuo old bean – but we know what you mean. Or do we? Most probably couldn’t tell you what the acronym stands for – but that doesn’t matter if they can explain that it’s the stuff (a ‘molecule’ would be better still!) that carries the information of inheritance and, as such, is responsible for all life. Go to the top of the class those who add that the code is in the form of chemicals called bases and there are just four of them (A, C, G & T). Something that simple doesn’t seem enough for all life but the secret is lies in the vast lengths of DNA involved. The human genome, for example, is made up of three billion letters.

A little bit of what is now history …

In the mid-1980s a number of scientists from around the world began to talk about the possibility of working out the sequence of letters that make up human DNA and thus identifying and mapping all the genes encoded by the human genome. From this emerged The Human Genome Project, a massive international collaboration, conceived in 1984 and completed in 2003. I quite often refer to this achievement as the ‘Greatest Revolution’ – meaning the biggest technical advance in the history of biology.

As that fantastic enterprise steadily advanced to its triumphant conclusion, it was accompanied by a series of mini-revolutions in technology that sky-rocketed the speed of sequencing and slashed the cost – the combined effect being an increase the efficiency of the whole process of more than 100 million-fold.

Brings us to the present …

These quite astonishing developments have continued since 2003 such that by 2009 it was possible to sequence 12 individuals in one study. By August 2016 groups from all over the world, coming together under the banner of The Exome Aggregation Consortium (ExAC), have raised the stakes 5,000-fold by sequencing no fewer than 60,706 individuals.

The name of the outfit tells you that there’s what you might think of as a very small swizz here: they didn’t sequence all the DNA, just the regions that code for proteins (exomes) – only about 1% of the three billion letters. But what highlights the power of current methods is not only the huge number of individuals sequenced but the depth of coverage – that is, the number of times each base (letter) in each individual exome was sequenced. In effect, it’s doing the same experiment so many times that errors are eliminated. Thus even genetic variants in just one person can be picked out.

seq-pic

Sequence variants between individuals. For most proteins the stretches of genomic DNA that encode their sequence  are split into regions called exons. All the expressed genes in a genome make up the exomeBy repeated sequencing The Exome Aggregation Consortium have shown that genetic variants in even one person can be reliably identified. Variants from the normal sequence found in four people are shown in red, bold letters.

It turns out that there are about 7.5 million variants and they pop up remarkably often – at one in every eight sites (bases). About half only occur once (which illustrates why DNA fingerprinting, aka DNA profiling, is so sensitive). As Jay Shendure put it, this gives us a “glimpse of the bottom of the well of genetic variation in humans.”

One of the major results of this study is that, by filtering out common variants from those associated with specific diseases, it will help to pin down the causes of Mendelian diseases (i.e. genetic disorders caused by change or alteration in a single gene, e.g., cystic fibrosis, haemophilia, sickle-cell anaemia, phenylketonuria). It’s clear that, over the next ten years, tens of millions of human genomes will be sequenced which will reveal the underlying causes of the thousands of genetic disorders.

The prize … and the puzzle

The technology is breathtaking, the amount of information being accumulated beyond comprehension. Needless to say, private enterprise has leapt on the bandwagon and you can now get your genome sequenced by, for example, 23andMe who offer “a personalised DNA service providing information and tools for individuals to learn about and explore their DNA. Find out if you are at risk for passing on an inherited condition, who you’re related to etc.” All for a mere $199!!

But you could say that the endpoint – the reason for grappling with DNA in the first place – is easy to see: eventually we will be able to define the molecular drivers of all genetic diseases and from that will follow ever improving methods of treatment and prevention.

Nevertheless, in that wonderful world I suspect we will still find ourselves brought up short by the underlying question: how one earth does DNA manage to carry the information necessary for all life?

For those who like to ponder such things, in the next piece we’ll try to help by looking at DNA from a different angle.

References

Ng, SB. et al. (2009). Nature 461, 272-276.

Lek, M. et al. (2016). Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291.

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Re-writing the Manual of Life

A little while ago we talked about a fantastic triumph by a team at Great Ormond Street Hospital (Gosh! Wonderful GOSH) in using a form of immunotherapy to save a little girl. What they did was to take the T cells from a sample of her blood and use gene editing – molecular cutting and pasting – to remove some genes and add others before growing more of the cells and then putting them back into the patient.

Gene editing – genetic engineering that removes or inserts sections of DNA – uses engineered nucleases, enzymes that snip DNA but do so in a controlled way by homing in on a specific site (i.e. a defined sequence of As, Cs, Gs and Ts).

We mentioned that there are four main ways of doing this kind of engineering – the GOSH group used ‘transcription activator-like effectors’ (TALEs). However, the method that has made the biggest headlines is called CRISPR/Cas, and it has been very much in the news because a legal battle is underway to determine who did what in its development and who, therefore, will be first in line for a Nobel Prize.

Fortunately we can ignore such base pursuits and look instead at where this technology might be taking us.

What is CRISPR/Cas?

CRISPRs (pronounced crispers) are bits of DNA that contain short repetitions of base sequence, each next to a ‘spacer’ sequence. The spacers have accumulated in bacteria as a defence mechanism – they’re part of the bacterial immune system – and they’re identical to sequences found in viruses that infect microbes. In other words, the cunning bugs pick up bits of dangerous viruses to make a rogues gallery so they can recognize and attack those viruses next time they pop in.

Close to CRISPR sit genes encoding Cas proteins (enzymes that cut DNA, so they’re ‘nucleases’). When the CRISPR-spacer DNA is read by the machinery of the cell to make RNA, the spacer regions stick to Cas proteins and the whole complex, including the viral sequences, can roam the cell seeking a virus with genetic material that matches the CRISPR RNA. The CRISPR RNA sticks to the virus and Cas chops its DNA – end of virus. So Cas, by binding to CRISPR RNA, becomes an RNA-guided DNA cutter.

crispr-pic

CRISPR-CAS: Bug defence against invaders. Viruses can attack bacteria just as they can human cells. Over time bugs have evolved a cunning defence strategy: they insert short bits of viral DNA into their own genome (above). These contain repeated sequences of bases and each is followed by short segments of ‘spacer DNA’ (above). This happens next to DNA that encodes Cas proteins so that both are ‘read’ to make RNA (transcription). Cas proteins bind to spacer RNA, leaving the adjacent viral RNA free to attach to any complementary viral DNA it encounters. The Cas enzyme is thus guided to DNA that it can cleave. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

Why is CRISPR/Cas in the headlines?

We saw in Gosh! Wonderful GOSH how the Great Ormond Street Hospital team tinkered with DNA and in Self Help – Part 2 we summarized another way of doing this using viruses (notably a disabled form of the human immunodeficiency virus) to carry novel genes into cells.

A further arm of immunotherapy attempts to reverse an effect called checkpoint blockade whereby the immune system response to tumours is damped down – e.g. by using antibodies that target a protein called PD-1 (Self Help – Part 1).

Now comes news of a Chinese trial which will be the first time cells modified using CRISPR–Cas9 gene editing have been injected into people. The chap in charge is Lu You from Sichuan University’s West China Hospital in Chengdu and the plan is to take T cells from the blood patients with metastatic non-small cell lung cancer for whom chemotherapy, radiation therapy and other treatments have failed.

The target will be the PD-1 gene, the idea being that, if you want to stop PD-1 doing its stuff, far better than mucking about with antibodies is to just knock out its gene: no gene no protein! What could possibly go wrong?

Well, wonderful though CRISPR is, it doesn’t always hit the right target but in this trial the cells can be tested to make sure it’s the PD-1 gene that’s been zonked – so that shouldn’t be a problem. However, it’s a blockbuster in that all the multiplied T cells put back into the patient will be active – i.e. will have lost the PD-1 brake. Whilst that may be good for zonking tumours, goodness knows what it might do elsewhere.

The initial trial is on a small scale – just 10 people. If there are problems one possibility is to try to take the T cells from the site of the tumour, which might select those specifically targeting the tumour – not straightforward as lung cancers are difficult to get at.

Anyone for a DNA upgrade?

It’s hard to say where all this is leading. However, as Chinese scientists have already made the first CRISPR-edited human embryos and the first CRISPR-edited monkeys, the only safe bet is that China will be to the fore.