An innovative technology that uses bacteriophage virus enzymes to "turn on and off" plant and animal genes of interest is the basis of a patent application recently filed by Embrapa. It allows, for example, that a plant exhibit resistance to drought only when, and if, it is subjected to water stress conditions. Bacteriophage viruses have the natural ability to infect bacteria. They adhere to the cell wall of these organisms, pierce it and inject their DNA. Observing this natural process, scientists realized that it would be possible to use these microorganisms as genetic tools to control gene expression. In addition, the fact that viruses are the most abundant biological group on the planet guarantees scientists vast amounts of genetic material to work with. Scientists also found that bacteriophage viruses, or phages, as they are also known, have the ability to integrate their own genome with that of bacteria. This process is mediated by enzymes called integrins, capable of integrating the genome of the virus with that of the host organism (bacteria). Functional biocircuits in different locations of the genomes The use of bacteriophage integrins as genetic tools for several applications has been a reality in biotechnology for over 25 years, as the main inventor of the technology, the Embrapa Genetic Resources and Biotechnology (DF) researcher Elibio Rech, explains. The novelty developed by the Brazilian team is to use six enzymes of the serine-integrase type in different locations of the genome, making the formation of functional biocircuits possible. According to the researcher, "integrins promote the breaking and binding of DNA sequences at specific points, resulting in accurate genetic rearrangements," he explains. This makes it possible to act precisely on the snippets of interest in the genetic code; for example, to silence a stretch that would express a genetic disease, or to cause a gene responsible for heat resistance to be expressed. "They are efficient and versatile tools for applications in experimental biology, biotechnology, biology and gene therapy," Rech says. The scientist explains that by introducing integrase into the DNA of a plant or animal, it is able to reverse the genetic sequence, preventing the chain of proteins from being recognized and, consequently, the gene being expressed. In informal language, it is able to "turn off" the function of that gene. When it is removed, the gene is expressed, that is, "reattached". Adaptation to drought, cancer control and pollution sensor A concrete example is the induction of tolerance to climatic stresses in plants, such as drought. According to the researcher, the technology opens the possibility of putting in the same product the integrases to "turn on and off" the expression of tolerance genes. "That is, when subjected to drought, the genes are activated, and, after the end of the season, deactivated," he adds. This is just one of the examples, but the applications may include gene expressions related to other climatic and environmental stresses, as well as resistance to diseases in plants and animals. "The most exciting and promising applications of bacteriophage virus integrins, particularly serine, are in the assembly of metabolic pathways and genetic circuits because they allow the reconstruction of whole metabolic pathways, by linking and deactivating specific genes in the genome," the researcher predicts. The tests that prove the efficacy of the methodology were done with six different types of integrases in plant cells (Arabidopsis thaliana protoplast), animals (bovine fibroblast) and human (tumor cells), and demonstrated a strong potential of application not only for agronomic research, but also for veterinary and human health research. In the latter case, one can foresee, for example, the use of a given integrase to interrupt the process of cell division of a cancerous tumor. Another example of using the methodology would be to control levels of water pollution, where the integrase would emit a "signal" when water reached a certain level of pathogenic bacteria that affects humans. Technology improves genomic editing by CRISPR Another contribution of patented knowledge is to support today's most promising technology in the field of genome editing, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Roughly, CRISPR works as a spellchecker, which allows to "cut" and "paste" genes of interest into the DNA of any species, without the inclusion of genes from other species (transgenic). Genome editing allows the development of pest-resistant crops, correcting defective genes in animals, and rewriting entire genomes of microorganisms. Despite its highly promising potential, some cases of non-target effects of this technology have been reported, especially with respect to the insertion of genes at random locations of the genome. According to Rech, the use of integrins in synergy with the enzymes that cut the genome, used in the CRISPR technology, allows the inactivation of these enzymes after the editing of genomes, avoiding undesirable effects of nonspecific cuts in the genome. In addition, it is possible to envisage the use of integrins as markers for insertion of synthetic chromosomes into the host genome with a further benefit: the potential to bind and deactivate genes within the chromosomes. For Rech, this is another step in scientific research to increase the technological domain in the generation of genetically modified organism characteristics man desires. "While single switches and genetic circuits are at an early stage of development, it is possible to predict near future, in which multiple switches become the norm, allowing more precise control of gene regulation and expression in plants and mammalian cells for the development of innovative processes and products for the benefit of humans and of the environment," the researcher believes. The technology that gave rise to the patent application was tested for almost 3 years at Embrapa Genetic Resources and Biotechnology laboratories in partnership with the University of Brasilia (UnB) and the Oswaldo Cruz Institute (Fiocruz). The inventors of Embrapa are the researchers Elíbio Rech, Andre Melro Murad, Leila Barros, Cristiano Lacorte, Eduardo de Oliveira Melo and Lilian Hasegawa Florentino. UnB’s participant is Cintia Coelho and the one from Fiocruz is Martin Bonamino. Synthetic biology: the imitation of life in the laboratory None of this would be possible without the mastery of synthetic biology, an area of science to which Rech has been working for years at Embrapa. "This is a strong ally of scientists in the generation of products derived from biotechnology and in its large-scale development, because it allows to ‘copy’ the processes of nature in the laboratory," he explains. The area integrates knowledge from different disciplines such as biology, chemistry, physics, mathematics, computer science, biotechnology and engineering to the projection and construction of new functions and biological systems generated in laboratories. The goal of synthetic biology is to create artificial life forms from natural elements. It breaks down the boundaries between the natural life and the manipulable technology with the objective of generating new products, technologies and applications. Translation: Ana Maranhão Nogueira