CRISPR/Cas against Huntington's Disease
Tuyana Malankhanova, Suren Zakiyan, "First-hand Science" No. 4(75), 2017
Today, according to the Harvard Center for Neuroscience, only in the USA about 5 million people suffer from Alzheimer's disease; 1 million from Parkinson's disease; 400 thousand from multiple sclerosis; 30 thousand each from amyotrophic lateral sclerosis (Lou Gehrig's disease) and Huntington's disease. These disorders, belonging to the group of neurodegenerative diseases, are poorly studied, and there are no effective treatment methods for them. A gene editing tool, the CRISPR/Cas9 system, which is currently actively and successfully used in biology and medicine, can help solve this problem.
Over the past decades, the average life expectancy of a person has increased, and now more people live to the age when various diseases associated with old age can begin to manifest themselves. Such "diseases of old age" include neurodegenerative diseases, most of which are characterized by the gradual and selective death of specific types of nerve cells. The causes of these diseases are most often disorders in the genome.
Consider this problem on the example of Huntington's disease (other names – Huntington's syndrome, Huntington's chorea or Huntington's), manifested by symptoms such as increasing loss of motor control and mental disorders. Despite the fact that the genetic mutation causing this disease was identified more than 20 years ago, the molecular mechanisms of its development are still not fully understood, as well as no effective therapy for this disease has been found.
People with Huntington's disease have abnormal and uncontrolled movements of almost all muscles of the body ("St. Vitus dance"), as well as mental and cognitive disorders. This serious disease usually begins to manifest itself after 35 years and has a progressive character.
Modeling the disease
The cause of Huntington's disease is an increase in the number of trinucleotide repeats of CAG in the HTT gene encoding the huntingtin protein. Normally, this gene contains from 8 to 36 repeats, but when this number reaches 37 or more, the development of the disease begins. The CAG triplet encodes the amino acid glutamine. Accordingly, Huntington's disease synthesizes huntingtin with an elongated polyglutamine chain. Such a protein has an irregular spatial structure and cannot perform its function in cells, which, by the way, is still unknown for sure. In addition, in the striatum, one of the parts of the brain that regulate muscle tone, a mutant protein forms aggregates that have a toxic effect. At the same time, the severity of the disease directly depends on the number of CAG repeats.
Above: the "healthy" HTT gene encoding the huntingtin protein. Bottom: mutant HTT gene
To investigate the molecular processes leading to the development of pathology, as well as to test new drugs, we need a biological model that reproduces human disease as closely as possible. To create a model of Huntington's disease, scientists introduced a mutant human HTT gene into the genome of laboratory mice. However, the results obtained in such "mouse" models do not always accurately reflect the processes occurring in the human body. The output is cell lines obtained from humans.
To study Huntington's disease, it is necessary to have striatum cells, but it is difficult to obtain such a biomaterial. This problem is solved with the help of induced pluripotent stem cells (iPSCs), which can be easily obtained from human skin or blood cells by reprogramming. Further, these cells can be grown in unlimited quantities and differentiated (transformed) into almost any desired type of cells.
The CRISPR/Cas9 genomic editing system is currently used to create cell lines simulating Huntington's disease through the production of induced pluripotent stem cells (iPSCs) from human somatic cells.
Usually, when modeling diseases, cells from sick people are used, and as a control – from a healthy donor. However, due to the presence of a huge number of single-nucleotide polymorphisms in the human genome, such pairs of cells are not quite correct to compare. The ideal control for "sick" cells will be cells with exactly the same genome, but without the mutation that causes the disease. Such cells are called isogenic pairs or lines. To create isogenic lines and use the CRISPR/Cas9 gene editing tool.
Exploring the gene environment
Despite the fact that Huntington's disease is a monogenic disease, i.e. caused by a mutation in one gene, there are more than 100 so-called modifier genes that can affect the time of the onset of the first symptoms, the severity of the disease, etc. The most convenient tool for elucidating the role of these genes in the pathogenesis of Huntington's disease is the CRISPR/Cas9-based GeCKO system (Shalem et al., 2014). It allows you to create entire libraries of gene targets for CRISPR/Cas9 and turn off thousands of genes in just a couple of steps. Thus, it is possible to knock out (turn off) all modifier genes in a short time and find out how this affects the viability of cells, the rate of their division, etc.
With the help of CRISPR/Cas9-based tools, it is now possible to edit not only the genome, i.e. hereditary information, but also the epigenome, which reflects changes in the work of genes that do not affect the structure of DNA. Thanks to genomic editing, we have the opportunity to change the expression level of the target genes. For this purpose, tools have already been developed that use "switched off" Cas9, various enzymes are attached to it that can activate or, conversely, suppress the level of production of a particular protein. For example, in the case of Huntington's disease, mutant protein synthesis can be specifically suppressed, thereby preventing the formation of protein aggregates and the death of striatum neurons.
However, it is also possible to activate compounds that will contribute to the accelerated removal or degradation of proteins with an irregular structure. For example, this effect is achieved by activating heat shock proteins (chaperones), which attach "death marks" to the wrong proteins. The proteins labeled in this way fall into special cellular structures, where they are destroyed.
In addition, CRISPR/Cas9–based epigenome modification tools are able to change the level of protein synthesis by modifying chromatin, a compact complex of DNA, RNA and proteins in chromosomes. It is possible to make such modifications to chromatin that will lead to a more "loose" DNA stacking, which activates transcription (reading genetic information on RNA). Other modifications may, on the contrary, contribute to a more compact DNA stacking and slow down or even completely block transcription.
Treating?
CRISPR/Cas9 can also be used to treat Huntington's disease. There are two options for exposure: shortening of CAG repeats or specific shutdown of the mutant copy of the gene.
In the first case, double-strand breaks are introduced into the sequence with repeats using CRISPR/Cas9. However, such an intervention has the opposite result – an increase in the number of CAG repeats. The problem was solved by using Cas9 nicases – mutant Cas9 proteins that introduce single-strand breaks into DNA. The experiment showed that consecutive single–strand breaks in regions with repetitions in the worst case do not change the length of the section with repetitions, and at best lead to its expected shortening (Cinesi et al., 2016).
The scheme of shortening the elongated path of repeats of the HTT gene CAG using the standard CRISPR/Cas9 system, which contributes to DNA double-strand breaks, and using Cas9 nicks that introduce single-strand breaks. By: (Cinesi et al., 2016).
The gene shutdown strategy can be used in those rare cases when there are single-nucleotide differences between two copies of the HTT gene. If such polymorphisms are located on both sides of the site with incorrect repeats, they can be used as targets for CRISPR/Cas9 scissors. In this case, the normal copy of the gene will not be affected (Shin et al., 2016).
Huntington's disease studies using the CRISPR/Cas9 system are currently being conducted in Of Russia. Thus, in the Laboratory of Epigenetics of Development of the Institute of Cytology and Genetics SB RAS (Novosibirsk), work is underway to obtain isogenic stem cell lines simulating Huntington's disease by introducing elongated CAG tracts into the normal HTT gene, as well as to differentiate iPSCs into striatum neurons. In the future, Novosibirsk researchers plan to move on to studying the role of modifier genes in the pathogenesis of this disease using a cellular model.
There is reason to hope that the research results will lead to the creation of an effective treatment method. In addition, they may help in the study of other neurodegenerative diseases, the development of which is caused by a similar mutation.
Literature
1. Medvedev S. P. How to edit heredity // Science at first hand. 2014. Vol. 55. No. 1. pp. 10-14.
2. Cinesi C., Aeschbach L., Yang B., Dion V. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase // Nat. Commun. 2016. V. 7. P. 13272–13281.
3. Shalem O., Sanjana N. E., Hartenian E. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells // Science. 2014. V. 343. N. 6166. P. 84–87.
4. Shin J. W., Kim K.-H., Chao M. J., et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9 // Hum. Mol. Genet. 2016. V. 25. N. 20. P. 4566–4576.
About the authors:
Tuyana Bayirovna Malankhanova is a post–graduate student of Novosibirsk State University, senior laboratory assistant at the Laboratory of Epigenetics of Development of the Institute of Cytology and Genetics SB RAS (Novosibirsk). Author and co-author of 3 scientific papers and 1 monograph.
Suren Minasovich Zakiyan – Doctor of Biological Sciences, Professor, Head of the Laboratory of Epigenetics of Development of the Institute of Cytology and Genetics, Stem Cell Laboratory of the Center for New Medical Technologies of the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk); Laboratory of Molecular and Cellular Medicine of the National Medical Research Center. Academician E. N. Meshalkina (Novosibirsk), Laboratory of Cellular Engineering of the Novosibirsk State University. Author and co-author of more than 200 scientific papers, including 33 monographs, and 5 patents.
Portal "Eternal youth" http://vechnayamolodost.ru