Modulating CRISPR-Cas genome editing using guide-complementary DNA oligonucleotides

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

Evaluation of miR-222 Expression in HBV Infected Patients in Comparison with HDV and HBV Co-infected Patients

Background: Liver disease is more severe in HDV+HBV co-infected patients than HBV infected patients which seems to be related to differences in the expression of genes and other factors such as MicroRNAs (miRNAs). The aim of this study was to investigate miR-222 expression in HBV infected patients in comparison with HDV+HBV co-infected patients. Methods: First, total RNA was extracted from the serum samples and then, complementary DNA (cDNA) was produced using cDNA synthesis kit. Finally, miR-222 gene expression was measured using U6 as the internal control by quantitative PCR (qPCR). Results: The level of miR-222 expression in HDV+HBV co-infected samples was significantly up regulated. The fold change of the miR-222 expression between two groups was 3.3 (95% CI; 0.011- 17.63) with p<0.001. Conclusion: The expression of miR-222 was higher in HBV+HDV co-infected patients than HBV infected patients. Further studies should be conducted to confirm whether miR-222 can be a biomarker for prognosis of severe liver diseases.

Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Label-Free Manner Using Micromechanical Sensors

Coronavirus (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as a deadly pandemic. The genomic analysis of SARS-CoV-2 is performed using a reverse transcription-polymerase chain reaction (RT-PCR) technique for identifying viral ribonucleic acid (RNA) in infected patients. However, the RT-PCR diagnostic technique is manually laborious and expensive; therefore, it is not readily accessible in every laboratory. Methodological simplification is crucial to combat the ongoing pandemic by introducing quick, efficient, and affordable diagnostic methods. Here, we report how microcantilever sensors offer promising opportunities for rapid COVID-19 detection. Our first attempt was to capture the single-stranded complementary DNA of SARS-CoV-2 through DNA hybridization. Therefore, the microcantilever surface was immobilized with an oligonucleotide probe and detected using complementary target DNA hybridization by a shift in microcantilever resonance frequency. Our results show that microcantilever sensors can discriminate between complementary and noncomplementary target DNA on a micro to nanoscale. Additionally, the microcantilever sensors’ aptitude toward partial complementary DNA determines their potential to identify new variants of coronavirus. Therefore, microcantilever sensing could be a vital tool in the effort to extinguish the spreading COVID-19 pandemic.

From Prebiotic Chemistry to Supramolecular Biomedical Materials: Exploring the Properties of Self-Assembling Nucleobase-Containing Peptides

Peptides and their synthetic analogs are a class of molecules with enormous relevance as therapeutics for their ability to interact with biomacromolecules like nucleic acids and proteins, potentially interfering with biological pathways often involved in the onset and progression of pathologies of high social impact. Nucleobase-bearing peptides (nucleopeptides) and pseudopeptides (PNAs) offer further interesting possibilities related to their nucleobase-decorated nature for diagnostic and therapeutic applications, thanks to their reported ability to target complementary DNA and RNA strands. In addition, these chimeric compounds are endowed with intriguing self-assembling properties, which are at the heart of their investigation as self-replicating materials in prebiotic chemistry, as well as their application as constituents of innovative drug delivery systems and, more generally, as novel nanomaterials to be employed in biomedicine. Herein we describe the properties of nucleopeptides, PNAs and related supramolecular systems, and summarize some of the most relevant applications of these systems.

DNA-tethered Polymersome Clusters as Nanotheranostic Platform

Nanotheranostics combine the use of nanomaterials and biologically active compounds to achieve diagnosis and treatment at the same time. To date, severe limitations compromise the use of nanotheranostic systems as potent nanomaterials are often incompatible with potent biomolecules. Herein we emphasize how a novel type of polymersome clusters loaded with active molecules can be optimized to obtain an efficient nanotheranostic platform. Polymersomes loaded with enzymes and specific dyes, respectively and exposing complementary DNA strands at their external surface formed clusters by means of DNA hybridization. We describe factors at the molecular level and other conditions that need to be optimized at each step of the cluster formation to favor theranostic efficiency.

Prospects Of Non-Coding Elements In Genomic Dna Based Gene Therapy

: Gene therapy has made significant development since the commencement of the first clinical trials a few decades ago and has remained a dynamic area of research regardless of obstacles such as immune response and insertional mutagenesis. Progression in various technologies like next-generation sequencing (NGS) and nanotechnology has established the importance of non-coding segments of a genome, thereby taking gene therapy to the next level. In this review, we have summarized the importance of non-coding elements, highlighting the advantages of using full-length genomic DNA loci (gDNA) compared to complementary DNA (cDNA) or minigene, currently used in gene therapy. The focus of this review is to provide an overview of the advances and the future of potential use of gDNA loci in gene therapy, expanding the therapeutic repertoire in molecular medicine.