Pros and cons of CRISPR
Biologists will discuss a method that changes reality
The international congress "CRISPR–2018" dedicated to this method of genomic editing opened yesterday in the technopark of Novosibirsk Akademgorodok.
The congress brought together about 360 scientists from the USA, France, Germany, North Korea, Turkey, Kazakhstan, Belarus and 16 Russian cities, including Moscow and St. Petersburg. At the opening of the scientific forum, Academician Valentin Nikolaevich Parmon, Chairman of the Siberian Branch of the Russian Academy of Sciences, addressed its participants: "We are at a very interesting historical period when a genuine renaissance of Russian science will take place. For the first time in many years, it has been placed among the most important national priorities, and what you are doing – genomic technologies – has been named one of the key areas where Russia should reach the world level." "The objects that we would like to see in Novosibirsk in the near future are a large center of genomic technologies and a biocenter closely connected with them," Valentin Parmon stressed, recalling the documents emanating from the head of state on the development of science in Siberia and Russia as a whole.
Academician Vsevolod Arsenyevich Tkachuk, Dean of the Faculty of Fundamental Medicine of Lomonosov Moscow State University, expressed a similar point of view: "Tectonic shifts are taking place in our direction of science. The CRISPR/Cas9 genomic editing method appeared just at a time when many dogmas of biology are being revised... CRISPR/Cas9 is not only a tool of cognition, it gives hope that we will master functional genomics… A new science is emerging before our eyes." Speaking about the points of growth of new scientific directions, the Moscow academician stressed: "On behalf of the President of the Russian Federation, there will be four new genomic centers in the country, and where to create one of them, if not in Novosibirsk?"
The first scientific report at the plenary session of the Congress was made by Candidate of Biological Sciences Sergey Petrovich Medvedev on behalf of the research team led by Doctor of Biological Sciences Suren Minasovich Zakiyan (FITC "Institute of Cytology and Genetics SB RAS"). The topic was the use of CRISPR‑mediated systems and genetically encoded biosensors to create and study cellular models of neurodegenerative diseases. "Due to the increase in the average age of the population in most developed countries, the proportion of patients with diagnoses such as Alzheimer's, Parkinson's, Huntington's, spinal muscular atrophy and others is growing sharply," said Sergey Medvedev. – Numerous studies have not yet led to the appearance of drugs that can be used effectively and safely enough in the treatment of neurodegenerative diseases. This is largely due to a lack of understanding of the molecular genetic mechanisms underlying pathogenesis, as well as the lack of models that allow obtaining not only qualitative, but also quantitative data on the processes occurring in the neurons of patients."
The researcher stressed the importance of studying the genetic factors of cellular abnormalities characteristic of neurodegenerative diseases: we are talking about increased apoptosis, mitochondrial dysfunction, oxidative stress, impaired protein utilization. Biosensor substances that signal about these disorders (ideally, visually and quantitatively) become a tool for searching for these disorders. "The use of genetically encoded biosensors allows us to study the dynamics of events occurring in a living cell in real time," says Sergey Medvedev."In addition, the combined use of technology for creating models based on induced pluripotent stem cells and biosensors allows us to study the molecular genetic mechanisms of diseases on relevant types of patient cells, in particular, on various types of neurons."
"Now we are actively working with bioinformatics, and there is hope that soon we will learn to count the reacting cells, and, accordingly, statistically evaluate the effectiveness of certain effects on them," suggested S.P. Medvedev. At the same time, answering questions from colleagues, he stressed that the question of the basics of a particular medical technology has not yet been raised – we are talking about obtaining fundamental knowledge. "Our models are intended only for in vitro research," the scientist emphasized. – Personally, I am against integrating transgenic cells anywhere without studying them in detail. Yes, this kind of work is being carried out in the world with an eye to Parkinson's disease, but, firstly, for this purpose, basic cells-prototypes of neurons are first obtained, and secondly, all experiments are carried out in laboratories equipped according to the GMP standard (The International GMP standard - good manufactured practice - includes an extensive range of indicators We do not have such conditions, which must be met by enterprises that produce certain products intended for humans – Ed.). The maximum available is testing on laboratory animals."
Dmitry Olegovich Zharkov, Head of the Laboratory of the Institute of Chemical Biology and Fundamental Medicine of the SB RAS, Scientific Director of the strategic academic unit "Synthetic Biology" of the NSU, Professor of the Russian Academy of Sciences, Doctor of Biological Sciences, made a plenary report on the problem of the accuracy and effectiveness of targeted nucleases of genomic editing. We are talking, first of all, about the Cas9 nuclease, the recognizer protein of the reconstructed gene. "Despite the unconditional progress in the field of genome editing of cultured cells, there are a number of limitations that do not allow the use of CRISPR/Cas9 fully," the scientist said. "These limitations include, in particular, the low efficiency of Cas9 delivery to certain cell types (in particular, pluripotent stem cells) and the resulting extremely low efficiency of editing by homologous recombination."
Dmitry Zharkov named the main reasons for this: "Firstly, the Cas9 molecule is very large, it is not very convenient to work with such in principle. Secondly, it is a slow protein... And, finally, the worst thing is that the use of Cas9 is far from as accurate as can be imagined from the enthusiastic press reports that scientists have designed a new kind of monkey or learned how to treat a new specific disease… The task of improving the accuracy of Cas9 is very acute." "Today's level is tolerable in laboratory experiments, as well as in agriculture," explained D.O. Zharkov. "But when we talk about medicine, the situation is unacceptable from all sides, including the economic one: in order to get the right number of cells with guaranteed accurate genome editing, its complete sequencing is required, which at current prices amounts to millions of dollars per patient."
"DNA is not a digital construct, it reads analogically, like a physical picture," Dmitry Zharkov concluded. – The information encoded in it depends not only on its sequence, but also on which protein and how it is used to read it. The evolution of reading proteins goes in the direction of reducing the probability of error in this or that choice. Finally, Cas9, in all likelihood, evolved differently from other DNA-recognizing proteins, that is, under conditions of recognition of a relatively large target on a small genome and cell volume. Therefore, when we turn to the human cell, we get the inevitable offtargeting effects. This does not mean that we should abandon Cas9: we need to look for more sophisticated methods and designs to increase the accuracy of this protein."
On the opening day of the CRISPR–2018 congress, a film was shown to mark the 30th anniversary of the Laboratory of Epigenetics of Development of ICIG SB RAS and the presentation of the second edition of the three-volume monograph "Editing of Genes and Genomes" with scientific articles by 37 authors. The Scientific Forum will continue working until September 14.
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