From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems.

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.

Design of Nucleic Acid Biosensors Based on CRISPR/Cas Systems and Reporter Split Proteins.

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.

[The use of SPECT-CT for visualization of sentinel lymph nodes and lymph drainage pathways in prostate cancer patients].

There was studied the use of SPECT-CT for visualization of sentinel lymph nodes and lymph drainage pathways in 35 patients with localized prostate cancer (cT1-T2 N0 M0). Sentinel lymph nodes were imaged in 31 (89%) patients. There were advantageously detected drainage pathways to exterior (77%) and internal iliac (58%) lymph nodes. Visceral (29%) lymph nodes (paraprostatic, paravesical and mesorectal) were less determined. There was established high informative value of SPECT-CT for imaging sentinel lymph nodes, determining their location as well as identification of lymph drainage pathways in patients with prostate cancer.

[Clinical value of transperineal saturation biopsy in patients with suspicious for prostate cancer].

The aim of the study was to evaluate clinical value of transperineal saturation biopsy (TPSB) in 52 patients with suspicious for prostate cancer (PCa): 31 - primary patients and 17 - men with non-effective transrectal biopsy. PCa was diagnosed in 31 of 52 (59,6%) patients. Focal lesions revealed in 6 (19,4%), multifocal - in another 25 (80,6%) cases. TPSB is very helpful in primary diagnosis and for therapy planning.

[High-dose brachytherapy for prostate cancer in real time using 192-IR (specifics of dosimetric planning)].

There were analyzed dosimetric plans obtained during the first session of HDR brachytherapy in 70 primary prostate cancer patients. Assessments were subjected to dosimetric parameters (V100, D90, D2cc, D10) obtained after implantation in the prostate needle-intrastats estimated before and after adjustment of contours of the prostate and surrounding organs at risk. It was showed that in most cases they were matched to the intended dosimetric parameters: V100 average--94,1% (V100 more than 90% in 97.2% of cases), D90 average--104,3% (D90 100% achieved in 95.7% of cases). In contrast, when using primary plan dosimetry without estimation of changing the geometry of the prostate and organs at risk in 38.6% patients V100 value was below 80%, in 41.4% patients--was in the range from 80% to 90%. In 24.3% patients index D90 did not exceed 80%, in 31.4% patients determined in the range from 80% to 90% and in 24.3% patients was close to 100%. In the absence of correction of contours of the urethra and the prostate in 18% patients the value of D10 for the urethra was higher border 115% and could increase to critical 189%.