In 2012, two research teams discovered that adaptive bacterial immunity could be used as a powerful tool in genome editing in mammalian cells (Jinek et al., 2012). This revolutionary system is known as the CRISPR/Cas system and has sparked one of the fastest growth areas of science in recent years. Literally thousands of publications have already used this system and you will hear a lot more about system in the future…
So what is the CRISPR/Cas9 system? CRIPSR is an acronym for “clustered regulatory interspaced short palindromic repeats”. It consists of a set of CRISRP-associated (Cas) genes interspaced by variable DNA sequences or DNA arrays that are derived from foreign sequences, usually similar to those found in bacterial pathogens and phages. When CRISPR is complexed with Cas endonuclease, target sequences are attacked to cause double-stranded breaks, which is the premise of the bacterial immune system.
So what’s all the excitement about? In short, the CRISPR/Cas system makes it far easier to carry out genome engineering, which is the process by which target genes in a genome can be precisely modified. In mammals, this can alter the function of cells or tissues, or even the whole organism. This means researchers have a new tool box to understand how genes and proteins work. Until now, this has been difficult. In humans, each cell contains around 3 billion base pairs of DNA, which encode around 20,000 genes. Yet major differences between individuals can be caused by just one base change. For potentially lethal conditions such as sudden cardiac death, there is just a single base pair change. However, with these new tools, stem cells can be isolated from patients carrying these mutations and the defect can be corrected with the CRISPR/Cas9 system. This ability means in the lab two parallel stem cell lines can be created, one with the disease and one without. This will allow better understanding of the disease state and offer the potential to develop new drugs, which is of interest to the pharmaceutical industry.
The next logical step of precise genome engineering is to move from dishes in the lab to patients in the clinic. Perhaps one of the diseases that is becoming best studied is acquired immune deficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV). The CRISPR/Cas system has been used to engineer HIV resistance into human cells (Leibman and Riley, 2015) or target the HIV genome for degradation, thereby preventing the spread of the disease (Khalili et al., 2015). These therapies are still very experimental and some way off reaching the clinic but offer a new opportunity to treat disease.
There have also been efforts to take the CRISPR/Cas system one step further. A Chinese group investigated whether mutations in the β-globin gene could be corrected in human embryos (Liang et al., 2015). A mutated copy of this gene can lead to β-thalassaemia, which is a potentially fatal blood disorder. The attempt was somewhat abortive because efficiency of correction was very low at 14%. Moreover, edited embryos were mosaic, which means not all the cells in the embryo were corrected. Finally, the level of off-target events was high, meaning that other genes, such as delta-globin gene, were changed and this was not the desired outcome. This research activity has been highly controversial, sparking calls for restraint in ‘acting God’ and not carrying out further experiments on human embryos. The view, particularly in the USA (Lanphier et al., 2015), is that;
“Genome editing in human embryos using current technologies could have unpredictable effects on future generations. This makes it dangerous and ethically unacceptable. Such research could be exploited for non-therapeutic modifications. We are concerned that a public outcry about such an ethical breach could hinder a promising area of therapeutic development, namely making genetic changes that cannot be inherited”
“The precise effects of genetic modification to an embryo may be impossible to know until after birth.”
Nevertheless, the same views were vocalized when Louise Brown, the first in vitro fertilization (IVF) baby, was born at Oldham General Hospital on 25 July 1978. Yet, 37 years on, there are now more than 5 million IVF babies.
Another potentially controversial aim of the CRISPR/Cas9 system is elimination of malaria (Gabrieli et al., 2014). According to the World Health Organisation, malaria kills a child every minute, with the mosquito as the main transmitter of the disease. There is debate in the community about eliminating an entire mosquito population. Theoretically, this could be achieved via the creation of genome edited mosquitoes, which would be released so that they could decimate the wild population. The idea here is that the CRISPR/Cas would be used to insert a DNA cutting enzyme, called I-PpoI, into mosquito genome. In normal reproduction, half of the sperm bear the X chromosome and will produce female offspring, and the other half bear the Y chromosome and generate male offspring. The I-PpoI enzyme works by cutting DNA of the X chromosome during production of sperm, which, in turn, leads to sexual imbalance and eventually eradication of mosquitos from the eco-system. One viewpoint is that mosquito extinction will not affect the ecological balance because these insects do not fill ecological niches that other species could not repopulate. The counterargument is can we be sure of this? Besides, do humans have the right to eliminate a species? Also, will eliminating the mosquito be the forerunner for other species, such as birds which transmit pandemic flu?
Whatever the views, the CRISPR/Cas system is here to stay and its use will increase exponentially. The question is, how will it be used in the future? Surely, this is something that will require considerable ethical debate in the community.
This blog article was co-authored by Minh Duc Hoang (The University of Nottingham) & Professor. Chris Denning (The University of Nottingham)
Gabrieli, P., Smidler, A., et al. 2014. Engineering the control of mosquito-borne infectious diseases. Genome Biology. 15, 535.
Jinek, M., Chylinski, K., et al. 2012. A programmable dual-rna-guided DNA endonuclease in adaptive bacterial immunity. Science. 337, 816-821.
Khalili, K., Kaminski, R., et al. 2015. Genome editing strategies: Potential tools for eradicating hiv-1/aids. Journal of Neurovirology. 21, 310-321.
Lanphier, E., Urnov, F., et al. 2015. Don’t edit the human germ line. Nature. 519, 410-411.
Leibman, R. S. & Riley, J. L. 2015. Engineering t cells to functionally cure hiv-1 infection. Molecular Therapy. 23, 1149-1159.
Liang, P. P., Xu, Y. W., et al. 2015. Crispr/cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell. 6, 363-372.