ИСТИНА

Detection of pathogens is an acute concern for the diagnosis of a broad spectrum of human, animal, and plant diseases, environmental monitoring, and food safety management. Highly sensitive, specific, fast, and easy-to-use diagnostic methods are required. As the “gold standard” PCR method requires expensive laboratory equipment and qualified personnel, other approaches should be developed. A promising approach is the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with high specificity under isothermal conditions. r work was dedicated to the design and study of the applicability of a biosensor system based on dCas9 proteins fused to the domains of a split-enzyme reporter system. Two dCas9 proteins can bind to the target DNA sequence at spatially close sites without cutting them. The split reporter enzyme is responsible for the detection of spatial colocalization of these protein complexes. We determined possible mutual orientations of two dCas9 proteins at the target loci of genomic DNA, which are favorable for the interaction of the attached domains of the reporter protein. The optimal distances between the DNA binding sites of dCas9 proteins in different orientations (“PAM-direct”, “PAM in”, and “PAM-out”) were calculated. The modeling was performed to evaluate the dependence of the system structure on the distance along the DNA between the binding sites of dCas9 proteins.