Bypass protection
According to Professor Norbert Reich from the University of California at Santa Barbara, human cells do not like it when something alien is introduced into their internal environment. In the process of evolution, they have developed a mechanism for "getting rid of garbage", providing isolation and cleavage of foreign proteins and other unnecessary biomolecules, pathogens and even damaged cellular structures. Therefore, the results obtained by specialists working in the fields of biotechnology, biopharmacology, as well as genomic research and clinical medicine, including with the CRISPR-Cas9 genome editing technology, are as good as they can effectively bypass this protective mechanism and introduce proteins into animal cells.
Researchers working under the guidance of Professor Reich managed to develop a method that solves this problem. Their proposed technology, the effectiveness of which, according to researchers, is 100-1000 times higher than that of existing methods, provides the user with full spatio-temporal control over the delivery of DNA-cutting proteins. In other words, it allows you to control the moment and place of release of these proteins with accuracy down to individual cells and their components.
One of the recent breakthroughs in biotechnology is the use of genome–editing proteins – "molecular scissors" such as CRISPR, Cas and, in this study, Cre - to search, cut and insert specific fragments of DNA sequences. Based on the protective mechanism used by bacteria to recognize the DNA of viruses attacking them and tag them for destruction, experts have developed a method for recognizing, cutting and binding sequences of nucleotide base pairs of different lengths using various proteins. The potential of this technology is huge and ranges from fundamental research on the study of the functions and identification of genes to gene therapy methods that allow to eliminate the genetic defects causing the disease at the cellular level.
The key components of the gene editing system proposed by the authors are near-infrared light and hollow gold nanospheres covered with reporter DNA chains (fluorescent with red light) and a chimeric protein consisting of Cre-recombinase and peptides penetrating into the cell. Nanospheres act as a carrier, and Cre-recombinase and chimeric protein act as a homing system that comes into play at the moment when the target cell triggers a mechanism for getting rid of debris.
After penetration into the cell, the entire system is inside the endosome, a membrane sac that isolates it and transports it through the cell. However, in this state, it is completely inactive. To release nanospheres, cells are exposed to pulsating near-infrared radiation, which effectively penetrates deep into tissues and is harmless to cells.
Under the influence of near-infrared light, a very interesting process occurs: gold nanospheres are excited and shed everything from their surface. At the same time, the formation of nanobubbles occurs, forcing the endosomes to open slightly and release the protein contents outside. The released proteins find their way to the cell nucleus and penetrate inside, where Cre-recombinase begins to act, cutting DNA and inserting its reporter sequences into the sections.
The in vitro experiments carried out by the authors demonstrated the effectiveness of the method. At the end of the incubation period and exposure to near-infrared radiation, the cells inside which the protein-coated nanospheres penetrated began to glow red due to a fluorescent protein embedded in the genome.
The authors explain that as a fundamental research tool, genome editing with spatio-temporal control allows you to do a separate experiment in each cell. By activating or inactivating the target gene, it allows real-time observation of changes in the behavior of the cell and its interaction with the environment.
In clinical practice, it will provide an opportunity to selectively modify cells expressing the gene responsible for the development of pathology without affecting healthy tissues. In addition, the ability to control the moment of activation of the DNA editing mechanism eliminates the long-term consequences associated with the use of modern methods, which often leave the active form of the DNA editing system inside the cell.
Article by Demosthenes P. Morales et al. Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light is published in the journal Small.
Evgenia Ryabtseva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of the University of California, Santa Barbara: Better Genome Editing