CRISPR/Cas9 is a gene editing system that was discovered in bacteria and is now one of the hottest topics in biomedicine. You probably saw the headlines referring to designer babies and the end of disease. As a general rule academics are not easily excited, but the hyperbolic headlines do capture some of the excitement CRISPR has precipitated in a number of fields. I thought I would leave the ethical considerations aside (there is also a heated, complicated legal battle for the patent) and instead focus on what CRISPR is and what it can do.
Before we ask what CRISPR is, we should probably spell out the acronym (be warned, it’s not very helpful). CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. What this refers to are small pieces of viral DNA stored in the bacteria genome which guide the Cas9 protein to the DNA of viruses when they try and infect the bacteria cell. The Cas9 protein cuts the DNA making a double-strand break in the DNA helix and killing the invading virus.
To use this system in humans, the guide sequence is changed to a sequence matching a target gene. The double strand break by Cas9 then triggers the cell to repair the break – there are 2 main types of DNA repair; non-homologous end joining (NHEJ) where the 2 ends either side of the break are essentially just stuck back together, and homology directed repair (HDR) which is much more accurate. If the cell uses NHEJ it is likely that the gene would be ‘knocked out’; it would no longer encode a functional protein because random bases may be incorporated, or bits of the sequence deleted. If the cell uses HDR, the cell will use a template copy of the gene (the other parental DNA or an artificially introduced template) to make sure the DNA sequence is corrected.
In diseases like Sickle Cell Anaemia, reversing one mutation in a single copy of a gene would be enough to effectively cure the disease. It’s estimated there are at least 5000 diseases like this meaning CRISPR’s potential is huge. One of the leading CRISPR companies has gained approval for a clinical trial to use CRISPR to treat sickle cell anaemia and a related condition – beta thalassemia – where CRISPR will be used to turn on the foetal haemoglobin gene in extracted blood stem cells. These will then be reinjected back into the patients (who will have undergone chemotherapy or radiation to destroy old blood stem cells) and the edited cells will be able to transport oxygen around the body much more efficiently and ameliorate the symptoms. (There are many other clinical trials planned for 2018, here is a good article talking about them).
One of main issues with CRISPR/Cas9 is getting the system into the cells of a living animal (the sickle cell treatment above relies on blood cell extraction) but it appears a breakthrough has been made last month. In this study CRISPR was injected into ear cells encapsulated in positively charged fat molecules and used to knock-out the mutant copy of the TnC1 gene that was causing deafness in mice. This significantly enhanced the survival of sound-sensitive ear cells and improved the hearing of the mice (eight weeks after the injection, the mice’s reaction to an abrupt 120-decibel noise was measured, for anyone pondering how hearing is measured in rodents). This study is also noteworthy as the mutated copy of the gene differed from normal by just one base pair; there are 3 billion bases in the human genome. This is a very exciting study, inching CRISPR closer to the clinic.
The applications of CRISPR/Cas9 are not limited to treating inheritable conditions but may also involve altering the environment to protect against infectious diseases. This technology, known as a ‘gene drive,’ brings another heap of ethical questions as essentially it is an attempt to rig evolution. In gene drives currently under development, CRISPR/Cas9 is used to introduce a gene that confers resistance to the malaria parasite – this would then, if left uninterrupted, progress through the population at a very slow pace. However, and this is the key concept, the introduced gene would be an autocatalytic insertional mutant – this means that the gene inserted would then cause the same change to occur on the other matching chromosome. As a result, the gene would spread through the population of mosquitoes rapidly, increasing the inheritance of the gene to ≈97%. Making more and more of the population unable to carry the malaria parasite until eventually, in theory, malaria is eradicated. There are also plans to use gene drives to improve crop yields, control invasive species and promote the survival of endangered species!
In my back-breaking 1342 page ‘Molecular Biology of the Cell’ textbook, CRISPR receives a meagre few pages. In future editions I expect there will be many more pages devoted to a technology that is changing the field, even creating new fields, faster than anyone expected. I can say with some certainty that this won’t be my last CRISPR blog and there will be many more alarmist Daily Mail headlines to look forward to in the next few years!
OOPS! Just after I had this article locked, loaded and ready to go, I came across a study that isn’t damning for CRISPR’s hopes but presents an obstacle in its path to the clinic. A study found that humans have antibodies ready to attack the Cas9 protein – this is known as a ‘pre-existing adaptive immune response”. It is due to Staphylococcus aureus (a common species of bacteria) previously infecting the body. The body – not realising it would hinder future gene editing technologies – has prepared for another exposure to this bacterium by synthesising specific antibodies. The implications for CRSIPR are not dire (there are a wide range of Cas proteins that are being explored and delivery systems capable of subverting the immune system) but amid the excitement this is perhaps a sobering moment. I can’t help but think of Orgel’s second rule: “evolution is smarter than you are”.