RNA-Targeting CRISPR–Cas Systems and Their Applications

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR–Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR–Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems

Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3' end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3' terminus by the appropriate PAM element. IMPORTANCE: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial communities and, consequently, the process of disease development and progression

Conjugates of Aminoglycosides with Stapled Peptides as a Way to Target Antibiotic-Resistant Bacteria

The misuse and overuse of antibiotics led to the development of bacterial resistance to existing aminoglycoside (AMG) antibiotics and limited their use. Consequently, there is a growing need to develop effective antimicrobials against multidrugresistant bacteria. To target resistant strains, we propose to combine 2-deoxystreptamine AMGs, neomycin (NEO) and amikacin (AMK), with a membrane-active antimicrobial peptide anoplin and its hydrocarbon stapled derivative. The AMG−peptide hybrids were conjugated using the click chemistry reaction in solution to obtain a non-cleavable triazole linker and by disulfide bridge formation on the resin to obtain a linker cleavable in the bacterial cytoplasm. Homo-dimers connected via disulfide bridges between the N-terminus thiol analogues of anoplin and hydrocarbon stapled anoplin were also synthesized. These hybrid compounds show a notable increase in antibacterial and bactericidal activity, as compared to the unconjugated ones or their combinations, against Gram-positive and Gram-negative bacteria, especially for the strains resistant to AMK or NEO. The conjugates and disulfide peptide dimers exhibit low hemolytic activity on sheep red blood erythrocytes

The Nucleocapsid Protein of Human Coronavirus NL63

<div><p>Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the <i>Coronavirinae</i> subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.</p></div

References to sequence and structural data on polypeptides used to design the expression constructs of HCoV-NL63 nucleocapsid N- and C- terminal domains.

<p>References to sequence and structural data on polypeptides used to design the expression constructs of HCoV-NL63 nucleocapsid N- and C- terminal domains.</p

Localization of the N protein in cells infected with HCoV-NL63.

<p>Three culture systems were used: 293T_ACE2⁺ cells, LLC-MK2 cells and fully differentiated human airway epithelial cultures. Single confocal planes are presented. Blue color denotes nuclei, while green represents localization of the HCoV-NL63 nucleocapsid protein. Top image in each set: scale bar corresponds to 40 μm; bottom image: scale bar corresponds to 5 μm.</p

Cell cycle analysis conducted on cells transfected with the NL63-N encoding mRNA or control, maxFP-green encoding mRNA.

<p>Analysis was performed using LLC-MK2 cells (<b>A</b>.) and 293T cells (<b>B</b>.). Nocodazole was used as a positive control, while non-viral mRNA and DMSO were used as negative controls.</p

N protein and CTD are able to form dimers.

<p>Molecular weight values for WT nucleocapsid protein and its domains, as determined using mass spectrometry. Experimental details are provided in the text.</p><p>N protein and CTD are able to form dimers.</p

Interaction of the CTD and NTD with RNA and DNA.

<p><b>(A)</b> RNA or DNA samples were pre-incubated with the protein and subsequently separated on the agarose gel. Shifts (the nucleoprotein complex does not leave the well in this case) observed in the lines containing RNA and DNA pre-incubated with the NTD suggest strong RNA-NTD and DNA-NTD interaction. <b>(B)</b> Electron microscopy images of the NTD in the absence and in the presence of RNA. Micrographs were prepared using scanning electron microscope JSM-5410.</p

DSC curves for the first heating scans for the complete NL63-N protein and its domains.

<p>NL63-N: complete NL63-N protein, NTD: N-terminal domain, CTD: C-terminal domain. Samples were suspended in 50 mM NH₄HCO₃, pH 7.8 and scanned at the rate of 1 K/min. Protein concentration was 1 mg/ml.</p