— Highly sensitive, specific, rapid, and easy-to-use diagnostic methods for the detection of nucleic acids of pathogens are required for the diagnosis of many human, animal, and plant diseases and environmental monitoring. The approaches based on the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with a high specificity under isothermal conditions are an alternative to the polymerase chain reaction method, which requires expensive laboratory equipment. The development of the methods for signal registration with the formation of a DNA/RNA/Cas9 protein complex is a separate bioengineering task. In this work, a design was developed and the applicability of a biosensor system based on the binding of two dCas9 proteins with target DNA sequences (without their cutting) and detection of their colocalization using reporter systems based on split enzymes was studied. Using the methods of molecular modeling, possible mutual positions of two dCas9 proteins at a detectable locus of genomic DNA, allowing the split enzyme domains attached to them to interact in an optimal way, were determined. The optimal distances on DNA between binding sites of dCas9 proteins in different orientations were determined, and the dependence of the complex structure on the distance between the binding sites of dCas9 proteins was modeled. Using the methods of bioinformatics, the genomes of a number of viruses (including SARS-CoV-2) were analyzed, and the presence of genomic loci unique to the species, allowing the possibility of landing pairs of dCas9 proteins in optimal positions, was demonstrated. The possibility of a combined use of dCas9 proteins from different bacteria to expand the spectrum of detected loci was analyzed. The results of the work indicate a fundamental possibility of the creation of highly specific nucleic acid biosensors based on a combination of CRISPR/Cas9 technologies and split enzymes.

In the last decade, the CRISPR-Cas technology has gained widespread popularity in different fields from genome editing and detecting specific DNA/RNA sequences to gene expression control. At the heart of this technology is the ability of CRISPR-Cas complexes to be programmed for targeting particular DNA loci, even when using catalytically inactive dCas-proteins. The repertoire of naturally derived and engineered dCas-proteins including fusion proteins presents a promising toolbox that can be used to construct functional synthetic genetic circuits. Rational genetic circuit design, apart from having practical relevance, is an important step towards a deeper quantitative understanding of the basic principles governing gene expression regulation and functioning of living organisms. In this minireview, we provide a succinct overview of the application of CRISPR-dCas-based systems in the emerging field of synthetic genetic circuit design. We discuss the diversity of dCas-based tools, their properties, and their application in different types of genetic circuits and outline challenges and further research directions in the field.

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At the center of the modern CRISPR/Cas genome editing revolution is the engineered ribonucleoprotein complex of the Cas9 protein with sgRNA that is able to bind and cleave DNA at specific loci. The targeted activity is conferred by the ability of the complex to bind to the DNA regions which are complementary to the parts of the sequence encoded by the sgRNA. Apart from DNA cleavage activity the selective binding activity of CRISPR/Cas complexes alone have been exploited in many technological applications to target certain compounds to specific DNA loci (e.g. for detecting certain DNA sequences, marking up certain portions of the genome with fluorescent labels, altering epigenetic state of the genetic loci, etc.). The most common version of the CRISPR/Cas editing system is currently based on the spCas9 protein obtained from S. pyogenes. However, search for other systems which are more effective and have less off-target activity is under way. Particularly, Cas proteins from other species including alternative CRISPR systems (e.g. recently characterized CasX system) combined by artificially engineered mutations can be attempted[1,2]. Hence, an effective in vitro assay to characterize the binding affinity of the Casbased ribonucleoprotein complex to its target DNA sequence is of high methodological importance. An effective way of measuring the biomolecular complex affinity can be based on using the FRET microscopy by monitoring the increase of the FRET signal as the two molecules labeled with a corresponding pair of fluorescent dyes bind together [3,4]. In this work by employing atomistic molecular simulations we show that such measurements are possible to study the binding of Cas9-sgRNA complex to its target DNA by attaching Cy3 and Cy5 labels to specific sites at DNA and RNA molecules. We simulate various attachment possibilities for optimal experimental designs.