Characterization of CoCas9 nuclease from Capnocytophaga ochracea.

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

[Type II CRISPR-Cas System Nucleases: A Pipeline for Prediction and In Vitro Characterization].

The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.

[Repair of Double-Stranded DNA Breaks Generated by CRISPR-Cas9 in Pseudomonas putida KT2440].

Pseudomonas putida KT2440 is a metabolically versatile bacterium with considerable promise as a chassis strain for production and degradation of complex organic compounds. Unlike most bacteria, P. putida KT2440 encodes the Ku and LigD proteins involved in Non-Homologous End Joining (NHEJ). This pathway of repair of double-strand breaks (DSBs) in DNA has an intrinsic mutagenic potential that could be exploited in combination with currently available genome editing tools that generate programmable DSBs. Here, we investigated the effect of removal or overproduction of NHEJ-associated P. putida KT2440 enzymes on mutations generated upon repair of Cas9-mediated DSBs with the double purpose of characterizing the NHEJ pathway and investigating how it functionally interacts with the current gold standard tool for gene editing. The results of our work shed light on non-templated mechanisms of DSB repair in P. putida KT2440, an information that will serve as foundation to expand the gene engineering toolbox for this important microorganism.

Diversity of CRISPR-Cas-Mediated Mechanisms of Adaptive Immunity in Prokaryotes and Their Application in Biotechnology.

CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology.

[Intensity of cardiac free-radicals processes and expression of glutathione peroxidase and glutathione reductase genes in rats with adrenaline].

The correlation between changes in activities of glutathione peroxidase and glutathione reductase in heart of rats during development of adrenaline myocarditis and intensity of free radical processes estimated by biochemiluminesce parameters and the content of lipoperoxidation products was demonstrated. The maximal increase of glutathione peroxidase and glutathione reductase activities (in 1.8 and 1.4 times accordingly) was observed t 24 h after the development of the pathological process; this coincided with the maximum intensity of prosesses of free radical oxidation. Using combination of reverse transcriptions with real-time polymerase chain reaction the cardiac mRNA levels of glutathione peroxidase and glutathione reductase genes were determined during the development of adrenaline myocarditis in rats. Analysis of expression of glutathione peroxidase and glutathione reductase genes showed, that the level of this transcripts demonstrated 2,8- and 7,3- increase in rats with adrenaline myocarditis, respectively. Obviously, overexpression of these enzymes can increase the resistance of cardiomyocites to oxidative stress.