Wednesday, September 9, 2015

The future is now! The rise of genome editing

It’s an exciting time to be a biologist! Every few years it seems like there is another significant technical breakthrough that allows biological research either to speed up exponentially, or to enter into areas that were previously inaccessible. In just the last decade or so we’ve seen the publication and digitisation of the human genome (without which most current life sciences work would be either impossible or impractical), the development of super-resolution microscopy (allowing us for the first time to see live biological processes on a truly molecular scale), the facilitation of DNA sequencing (making it economical on a large scale), and the invention or improvement of a whole range of technologies (enzyme-conjugation systems, flow cytometry, fluorescence-activated cell sorting etc.) that won’t mean much to anyone outside the field but that have revolutionised the way research is done. It’s been a long road, but it finally seems like the ambitions of researchers are starting to be matched by the available technology, whether it be computational, mechanical, chemical, or biological. The latest innovation that is taking the biology world by storm is the enormous progress that has recently been made in an area that has incalculable potential in both academic and clinical contexts: genome editing. In this post I will try to explain these recent advancements, why researchers are excited, and why you should be too!

What is genome editing?

Genome editing is pretty much what you’d expect from the name; editing the DNA sequence within the genome of a particular cell. This can involve adding DNA, removing DNA, swapping some DNA for other DNA, or moving DNA around within the genome. It is difficult to overstate how powerful a tool genome editing can be when it comes to biological research. Much of the work done in molecular life sciences is trying to work out how various molecules fit into the whole machine that is an organism – genome editing allows researchers to directly tinker with these molecules (typically proteins, which are of course encoded by a their associated DNA sequence) and observe the effects. This could involve removing the gene encoding a given protein from an organism and seeing what defects arise. Alternatively, you could introduce a specific mutation in a gene to see if that has functional relevance, or introduce DNA encoding fluorescent marker proteins into the end of your protein of interest to see where it goes and what it’s up to. Genome editing elevates researchers from the level of pure observers into direct manipulators of a system.

Sounds great, what’s the catch?

The problem with genome editing until the very recent past was that it was extremely labour intensive and time-consuming. Possibly the simplest form of genome editing is to delete a gene from a cell. Had I wanted to permanently delete a gene from a cell just a few years ago, I would have typically have used a form of ‘site-specific recombinase’ technology that makes use of the natural process of DNA recombination to disrupt a specific gene. This is a long process and is only effective for use in whole organisms as recombination only occurs during the production of sperm and egg cells, not in normal dividing cells. So, I would need to perform my site-specific recombination in a mouse egg then implant it in a mouse and hope that I could get my ‘knock-out’ (as such organisms are known) mouse at the end. This has several obvious limitations: it’s a long process, it’s not particularly efficient, it involves the use of live animals, it can’t be done for human cells, it still means I have to harvest my desired cells from the mouse at the end, and it may not work at all if the gene deletion that I’ve made somehow messes up the development of the mouse beyond the foetal stage.

Bringing gene targeting to the masses

What was needed was a way to target genes directly within cells that you already had in your incubator, without the need to go around the houses making transgenic mice.  Some technologies were successfully developed that did achieve this goal, such as ZFNs and TALENs, but these involved the generation of a new protein each time you wanted to target a different gene. Designing new proteins is considerably harder and less predictable than just making a new sequence of DNA, so these approaches are only really used by specialists in genome editing. The first shimmer of hope that non-specialist researchers might get in on the direct manipulation of gene activity came in the late 1990s when the technique of RNA interference (RNAi) was successfully developed. RNAi doesn’t actually edit the genome of your target cell, but it does the next best thing. DNA in the genome only mediates its effects once it is transcribed into RNA and then translated into protein. RNAi takes advantage of a natural defence mechanism of eukaryotic cells against viruses to trick the cell into thinking that some of its own RNA comes from a virus. This means that the cell will specifically destroy that RNA, and so the DNA from which it was transcribed is effectively inactive. RNAi is simple to design as it only involves the inserting into your cells some double-stranded RNA with the same sequence that you want to disrupt, rather than designing a new protein every time. You can make the effects permanent by inserting DNA into the genome of the cell that encodes the specific double-stranded RNA so there’s no need to keep adding more, the cell does it for you. RNAi was a sensation and was rapidly adopted by researchers all across life sciences, earning its inventors the 2006 Nobel Prize in Medicine.

Revolutionary though RNAi undoubtedly was, it still came with a long list of caveats. Firstly, it wasn’t a true ‘knock-out’ – some of the target RNA would always make it through unscathed and so there would still be low levels of the protein you wanted to get rid of. For this reason, RNAi-treated cells are typically termed ‘knock-down’ rather than ‘knock-out’. Secondly, it could have off-target effects since genes with similar sequences to the one you wanted to disrupt may also be affected. Thirdly, it was still difficult to make the knock-down truly permanent – cells would often slowly recover expression of the target protein. Fourth, it was only good for knocking out gene function – inserting selective mutations and other more subtle editing was not possible. Finally, it proved very tricky to do this in whole organisms. It was great for simple organisms like yeast or even larger model animals like nematodes and fruit flies, but putting it into mammals proved to be a significant hurdle. 

Going beyond RNAi

The ideal approach would be one that had the ease of use of RNAi but that would deliver an absolute, permanent change to the genome, could be used to engineer whole organisms, and would allow insertions and mutations as well as basic deletions. Given my enthusiastic introduction to this post you’ve probably guessed that such an approach has, at last, arrived. It’s informally known as CRISPR (pronounced ‘crisper’), which stands for clustered regularly spaced palindromic repeats, but the full name for the technology is the CRISPR/Cas system.

CRISPR is, in my humble opinion, fantastic! It works by using a guide RNA sequence (which you customise to target your DNA sequence of interest) to direct a bacterial protein (Cas9) to cut the genome only at a specific point in your target gene. The cell then tries to repair it in one of two ways: non-homologous end-joining (NHEJ) or homology-directedrepair (HDR). If you want to knock out a gene, you let NHEJ do its work. If this correctly repairs the cut then the Cas9 can just cut it again, over and over until it makes a mistake, which it will eventually. Once the mistake is made, your gene will be ruined and its sequence will no longer match the guide RNA so will stop getting cut by the Cas9. For more subtle modifications, you can insert a separate segment of DNA into the cell, which will then be used in HDR as a template for repairing the cut DNA. The repaired section will then contain the sequence that you included in the extra DNA, which could be a mutation, a tag, or whatever else.

The principle of CRISPR: guide RNA (gRNA) directs the cutting
of a target DNA sequence to allow either NHEJ or HDR.

Like RNAi, CRISPR takes advantage of natural defence mechanisms to achieve its effects. The normal job of Cas9 is to cut any foreign DNA that finds its way into bacteria, from viruses for example, thereby helping to defend bacteria from infection. It was originally discovered in 2007 by researchers at a Danish food company trying to make better virus-resistant bacteria for food production, but it quickly became evident that it could be much more significant. It only took 5 years to go from this initial discovery to its successful use in genome editing in cultured human cells. Since then the field has exploded, with CRISPR being used to generate living animals with edited genomes in a range of species including flies, fish, and mice, as well as non-viable embryos of primates and even humans (anyone interested in a more detailed story of how CRISPR was developed and how it works can find an excellent article in Science here.

Alongside such more ambitious work, CRISPR is rapidly becoming an everyday technique for researchers, myself included. It is now straightforward to generate edited cell lines to suit your research question, which opens up avenues of work that were, until recently, inaccessible. This has been facilitated by the fact that the CRISPR technology is freely and cheaply available to any lab that wants it – I bought all of the materials I needed to start using CRISPR for just 65 US$. This is to the credit of those who have had the greatest contribution to its development, as such an important technology could easily have been exploited for commercial gain. The scope of the technology has expanded rapidly, with new versions of the system popping up every couple of months with increased efficiency or specificity for one application or another. One exciting new option is to buy (for about 400 US$) a library of CRISPR guide RNAs that target every gene in the human genome individually. You can then expose millions of cells to these, look for cells exhibiting a specific effect of interest, and then sequence the genomes of those cells to see which genes were disrupted to cause the effect. What once would have taken years or decades to work out can now be done in a few months. It’s remarkable.

Where next for CRISPR?

The seemingly instantaneous appearance, development, and adoption of CRISPR has left a lot of people in the life sciences a little stunned and many people are still getting to grips with the brave new world that it has opened up. It seems certain now that CRISPR will very soon be used as routinely as many other molecular biology techniques. The real question, is what is the limit of CRIPR’s potential in the field of clinical science, and should we impose one? Clearly, something that is able to edit genomes has enormous potential when it comes to genetic disease. It is already possible to use CRISPR to alter genes in mice at the stage offertilisation, using a technique called intracytoplasmic sperm injection that is already widely used during IVF in humans. Soon it will be technically feasible to correct mutations in the genes of children conceived by carriers of genetic abnormalities. In principle this would give humanity the potential to slowly eradicate all single-gene genetic disorders, since the correction would be maintained in all of the descendents of the original child – a so-called germline correction.

Whilst this is an exciting possibility, it is one that has caused significant concern within the scientific community and beyond. It has been the general consensus for half a century that germline editing was a step too far as the stakes (i.e. the integrity of human genetics) are just too high. The UNESCO UniversalDeclaration on the Human Genome and Human Rights, for example, flatly states that germline editing could be “contrary to human dignity”. The constancy of this viewpoint was supported in part by the fact that germline editing was simply not possible and so fairly easy to write off, but that has now changed and so the debate is alive again. The emergence of CRISPR as a viable tool for germline editing has sparked a wave of cautionary remarks from geneticists and clinicians, including from the co-developers of CRISPR, demanding that steps be taken now to prevent any viable germline intervention in future. Indeed, some researchers have suggested that human germline cells in general should be off-limits (even if not used to make a viable human) because the public and/or policy makers may not adequately distinguish between this and non-germline editing and so lead to a harmful backlash against all genome editing.

Personally, I don’t have any objection to the principle of human germline editing, however I feel that the consequences for genomic integrity have to be much, much better understood before such approaches could begin to be applied. The explosion in the use of CRISPR in labs has understandably made some people nervous that the path towards germline editing will be similarly supersonic, however I don’t think we are in danger of that. Everyone involved in genome editing is aware of the issues, and few areas of molecular biology are under more public and political scrutiny than human genetic modification, so I don’t envisage any steps being taken along that path without extreme caution on all sides. Setting over-zealous policies into stone at this stage could be highly detrimental to future research that would have to labour under the yolk of rules from a more ignorant age. Having said that, we have to acknowledge that any technology can be abused and if we do successfully develop safe human germline editing there is probably not a lot we can do to prevent it being used for private or political ends. Is working to develop this technology whilst demanding that it only be used to cure disease a bit like if original researchers into nuclear fission had naively demanded that their work was only to be used in power plants and not bombs? If so, is it better to just not develop it in the first place? There is possibly an argument for this, but I don’t think so – sooner or later this work will get done by someone and I think it is best that it is done in full public view with appropriate scrutiny and debate. We can only have faith in humanity that it will not be abused.

In the meantime, though, those of us with more humble goals are enjoying the possibilities that CRISPR has opened up for us. I can honestly envision that almost every paper I publish in the future could involve CRISPR in one way or another. This means that all of molecular biology will be lifted, so the pace of research will quicken, and new drugs, therapies, or just basic understanding will be faster to develop. These are, as I say, exciting times!

1 comment:

  1. I hope all is well with you. I'm doing my best to cope but it's bad man, so bad, to slog my way over to your place just telling myself you've bound to have posted again by now. Unless you're dead or something, but that's treatable now so you should be able to post if you care enough.