CRISPR/Cas9 System could help in the discovery of new drugs and in the prevention of infectious diseases

CRISPR/Cas9 could help accelerating investigations on genetic modifications in disease-transmitting insect vectors to create insects without efficiency as vectors

Picture a system able to precisely edit genomic DNA in cells and organisms and still promote strong implications in basic research, medicine and biotechnology. An efficient, practical and simple tool able to work in large-scale on genomes and carry a significant acceleration in genetic function analysis processes. It exists and is already a reality in Brazil. It is called the CRISPR/Cas9 System.

This new tool can help in the discovery of new drugs or improve the prevention of innate or acquired genetic diseases or even those caused by infectious agents. The CRISPR/Cas9 technology could also contribute to the enhancement of investigations related to genetic modifications in disease-transmitting insect-vectors. The system represents a great ease in gene edition processes in various organisms and their outcomes are important for pure knowledge, basic science and can also open new horizons to applied science.

To know more about the theme, the press advisory at the Brazilian Society of Tropical Medicine talked to full professor in Molecular Parasitology at Ribeirão Preto Medical School, University of Sao Paulo, Angela Kaysel Cruz, who in 2017, started the first knockout experiments in different Leishmania species.

CRISPR-Cas9 Confers Adaptive Immunity. Left Panel: After a phage has injected its DNA into the bacterial cytoplasm, a protein complex forms consisting of Cas1 and Cas2. A sample sequence, termed protospacer, is acquired from the phage DNA by the Cas1-Cas2 complex (1). This sequence is then integrated into the CRISPR genetic locus (2). The sequence is now called a spacer. Several spacers from earlier contacts with phages are stored in the CRISPR genetic locus. Right Panel: Spacer sequences are transcribed as RNA, and Cas9 protein is synthesized (1). RNA binds to Cas9 to form a surveillance and interference complex (2). The complex then scans intracellular DNA for matching sequences (3). If a matching phage sequence is found, Cas9 destroys the DNA by cleavage (4). Source: https://goo.gl/MPJA5F

BSTM: Dr. Angela, tell us about your experience with CRISPR/Cas9. How and when did you begin working in this field?

Dr. Angela: I work with species of protozoa from the Leishmania genus. Generally, tools for genetic manipulation of these parasites have to be re-engineered, re-thought and tested; since its genetic, structural and functional organization are different from most eukaryotes. This way, all new technologies generated in other organisms are then rearranged to trypanosomatids. Two research groups have published the first papers applying the CRISPR/Cas9 system to Leishmania in 2015 (groups from Y. Sterkers and G. Matlashewski, in France and Canada respectively). In 2016, we began developing our first experiments in my laboratory aiming to replicate the system and guidelines developed by the Canadian group, without success, until in 2017, when Eva Gluenzs group published a paper about the development of alternative tools and guidelines. The researcher supplied the necessary molecular constructions, and still in 2017, we started our first gene knockout experiments in different Leishmania species. Since then we have succeeded generating knockout for several genes of Leishmania major and Leishmania braziliensis. What is most impressive about the system is its efficiency and convenience. The speed in which the desired edition is achieved and the possibility to work in large-scale on genomes allows us to predict a significant acceleration in the genomic function analysis processes in large scale with important practical application outcomes.

BSTM: How does the CRISPR/Cas9 system work?

Dr. Angela: Genome edition by CAS9 enzyme (CRISPR-associated gene 9), an endonuclease then recognizes clustered regularly interspaced short palindromic repeats (CRISPR), is a transforming technology since Cas9 precisely and sequence-specifically cleaves the double DNA strand. A guide RNA (sgRNA) drives this specificity. The DNA repair, that occurs to restore the double-strand cleavage, allows the target sequence to be removed from the genome or precisely modified. This ability to precisely – and, methodologically hassle-free – edit genomic DNA in cells and organisms has marking implications for basic research, medicine and biotechnology.

BSTM: How can this new tool help preventing tropical diseases?

Dr. Angela: As I tried to explain earlier, the greatest advantage about this new tool is its simplicity and efficiency. I think CRISPR/Cas9 is a powerful catalyst for upcoming knowledge on the role of a gene or a set of genes operating an organism (or pathogen) or even its interaction with the host. Taking for example the several Leishmania species, we know there are multiple and redundant factors involved in the pathogenesis or virulence, and what this new tool can do is to contribute with information about these factors by easing the procedures of genetic parasite manipulation. The second phase, after editions, is to understand its effects, what can be very complex.

BSTM: Do you believe the CRISPR/Cas9 technology will be accessible to the poorest countries?

Dr. Angela: This technology is carried out in laboratories with stable infrastructure and funding conditions, in which experiments and genetic analysis can be performed. The following question, more difficult and that escapes our reach, is to apply these advances, eased by CRISPR/Cas9, to benefit the society in a way to include the poorest countries and/or marginalized populations.

BSTM: Using these new methods should revolutionize the development of more advanced tools to control tropical diseases. How will this affect the pharmaceutical industry in the production of new drugs and vaccines against tropical diseases?

Dr. Angela: I think the CRISPR/Cas9 System is a very powerful and useful tool that can be used in the most diverse organisms. This tool enables to accelerate knowledge generation about genes and their functions. This way, it is possible to predict that efficiently generating mutant parasites in large scale, as allowed by CRISPR/Cas9 edition, could ease processes of compound scans to discover new drugs or facilitate the development of genetically modified parasite lineages unable to cause the disease, but able to strike a long-lasting immune response, thus becoming vaccination lineages.

BSTM: Which progresses are expected with CRISPR/Cas9?

Dr. Angela: The most impacting use for the CRISPR/Cas9 so far, for its power and possible outcomes, was to edit genes in human embryos; in this field, we can find our highest expectations and the greatest dangers. We must understand that CRISPR is making it much easier to modify microorganisms, animals and plants. This calls for new regulations that scientists, development agencies and government regulatory agencies will soon face.

BSMT: Does CRISPR/Cas9 have limits? Which?

Dr. Angela: Yet a very powerful tool as already mentioned, there are still unknown issues – even for the recent development – or controversies. One of them refers to possible non-planned edition off-target events, what could represent, in some cases, an important limitation to the system.

BSTM: What can we expect from agricultural productivity with this technology?

Dr. Angela: Plant genome manipulation using the CRISPR/Cas9 system is already a reality and can have great impact outcomes and applications, especially due to the systems efficiency and range. However, I prefer make no statements about a knowledge field different from my own.

BSTM: The Aedes aegypti mosquito has been the worlds great villain in the last years. How can mutations in this vector help humanity win this war?

Dr. Angela: Genetic modifications in disease vectors have been implemented in research laboratories aiming a practical application and there are ongoing field essays. What the CRISPR/Cas9 could do is to contribute to speeding up the investigations, as I mentioned, for its efficiency and technical simplicity.

BSTM: Which are the ethical concerns regarding CRISPR/Cas9?

Dr. Angela: Ethical concerns permeate scientific investigations and all human activities. I will bring up the discovery of radioactivity by Marie Curie, over a hundred years ago. When M. Curie performed her first investigations on radioactivity and isolated radium for the first time, she did not have in mind the practical applications of her finding, she was performing pure science. The benefits and advances humanity achieved by the many practical applications, thanks to Maria Curies discoveries, are unquestionable. However, associated to these benefits, there are also huge prejudices and damages, such as the use of radioactivity knowledge by the weapon industry. The pernicious use does not diminish the importance of the benefits and I think this kind of consideration and reflection should permeate each and every scientific activity.

This way, we can extend these issues to a large number of other investigations and discoveries, among which, the CRISPR/Cas9 system and its use and optimization as a genome edition tool. There are numerous benefits, but there is also a possibility of its use to amoral or anti-ethical means. A strict and internationally agreed regulation on the uses of CRISPR/Cas9 is urgent and the first international conference to discuss science and human gene edition ethics took place in 2015. The attention by the international scientific community is mandatory to regulate the systems application within strict ethical precepts.

BSTM: Is it possible to reach a super-human, resistant to all infections?

Dr. Angela: We still have much to understand about the processes and relations between infectious agents and hosts before considering a resistant super-human, as you say. The CRISPR/Cas9 system represents a major facilitator in the process of gene edition in the most diverse organisms, but it is necessary to understand the genes role, their inter-relations and functions before modifying them. We must first learn what we need and what we should change.

BSTM: Will Eugenia become a reality?

Dr. Angela: We are now back to the ethical issues I mentioned above. Eugenia fits this discussion and demands great reflection and depth. I would rather not dive into this discussion right now.

BSTM: Would you like to add anything you may find important and that was not approached above?

Dr. Angela: I see the CRISPR/Cas9 system as a very powerful and useful tool, which can be used in the most diverse organisms – from unicellular to plants and humanity. It has allowed speeding knowledge about genes and their functions and this already has major outcomes for pure knowledge, basic science and can open new horizons for applied science. It can ease the discovery of new drugs or the prevention of innate or acquired genetic diseases or even diseases caused by infectious agents. However, we must not lose sight of what we still have to learn about potential weaknesses or limitations to the CRISPR/Cas9 system for its use in benefit of the society.…