The discovery of the CRISPR/Cas9 microbial adaptive immune system has revolutionized the field of genetics, by greatly enhancing the capacity for genome editing. CRISPR/Cas9-based editing starts with DNA breaks (or other lesions) predominantly at target sites and, unfortunately, at off-target genome sites. DNA repair systems differing in accuracy participate in establishing desired genetic changes but also introduce unwanted mutations, that may lead to hereditary, oncological, and other diseases. New approaches to alleviate the risks associated with genome editing include attenuating the off-target activity of editing complex through the use of modified forms of Cas9 nuclease and single guide RNA (sgRNA), improving delivery methods for sgRNA/Cas9 complex, and directing DNA lesions caused by the sgRNA/Cas9 to non-mutagenic repair pathways. Here, we have described CRISPR/Cas9 as a new powerful mutagenic factor, discussed its mutagenic properties, and reviewed factors influencing the mutagenic activity of CRISPR/Cas9.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6b^rd1 mice (rd1), which carry a spontaneous nonsense mutation in the pde6b gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through pde6b gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

The RNA-guided Cas12a nuclease forms a complex with a CRISPR RNA (crRNA) to cleave the double-stranded DNA target. Among others, Cas12a protein from Lachnospiraceae bacterium (LbCas12a) is widely used for biomedical research. For target recognition, LbCas12a requires a specific nucleotide sequence, named a protospacer adjacent motif (PAM). Besides the canonical TTTV PAM, LbCas12a can recognize other suboptimal PAMs. We examined a novel TTAA PAM for the LbCas12a nuclease and found that the specificity of cleavage was increased. We found that single nucleotide substitutions at all positions of the guide RNA except the 20th position blocked the cleavage of the target DNA. The type of nucleotide substitutions (U-A, U-C or U-G) did not affect the efficiency of cleavage in the 20th position. When we used the canonical PAM under the same conditions, we observed the cleavage of target DNA by LbCas12a in many positions, showing less specificity in given conditions. The efficiency and specificity of the LbCas12a nuclease were evaluated both by gel-electrophoresis and using FAM-labeled single-stranded probes. We were able to assess the change in fluorescence intensity only for several variants of guide RNAs. High specificity allows us to type single nucleotide substitutions and small deletions/insertions (1–2 nucleotides) and look for target mutations when knocking out.

Amyloids are the fibrillar protein aggregates with cross-β structure. Traditionally amyloids were associated with pathology, however, nowadays more data is emerging about functional amyloids playing essential roles in cellular processes. We conducted screening for functional amyloids in rat brain. One of the identified proteins was STXBP1 taking part in vesicular transport and neurotransmitter secretion. Using SDD-AGE and protein fractionation we found out that STXBP1 forms small detergent-insoluble aggregates in rat brain. With immunoprecipitation analysis and C-DAG system, we showed that STXBP1 forms amyloid-like fibrils. Thus, STXBP1 demonstrates amyloid properties in rat brain and in bacterial expression system.

Injury of the knee joint can lead to a range of adverse outcomes and significantly contributes to the development of the knee osteoarthritis. Currently, autologous platelet-rich plasma is used as a promising and safe method of treating osteoarthritis. Such plasma contains various growth factors, some of which are secreted after platelet activation. These factors can trigger a regenerative response and improve the metabolic functions of damaged structures. However, there are different protocols for preparing platelet-rich plasma, which results in preparations with different amounts of bioactive substances. As a result, the data obtained on the effect of platelet-rich plasma on the restoration of hyaline cartilage of the knee joint are very contradictory. A search for publications on a given topic was performed in the eLIBRARY, PubMed (MEDLINE), Ovid, Science Direct, Google Scholar databases, and also a search was conducted for clinical trial data on the treatment of knee osteoarthritis with platelet-rich plasma over the past 20 years. Publications dealing with other aspects of the application of this technology were excluded from the search results. An analysis of published clinical trial results found that, in most cases, patients treated with platelet-rich plasma reported improved pain and joint function, with only three studies showing no difference between platelet-rich plasma and placebo. Thus, this technology is generally promising for use in the treatment of knee osteoarthritis, however, methods of obtaining and activating platelet-rich plasma, as well as the age and comorbidities of the patient, may affect the effectiveness of therapy.

CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease fromLachnospiraceaebacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNAin vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice. In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice. This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)