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Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins offer a breakthrough platform for cheap, programmable, and effective sequence-specific DNA targeting. The CRISPR-Cas system is naturally equipped for targeted DNA cutting through its native nuclease activity. As such, groups researching a broad spectrum of biological organisms have quickly adopted the technology with groundbreaking applications to genomic sequence editing in over 20 different species. However, the biological code of life is not only encoded in genetics but also in epigenetics as well. While genetic sequence editing is a powerful ability, we must also be able to edit and regulate transcriptional and epigenetic code. Taking inspiration from work on earlier sequence-specific targeting technologies such as zinc fingers (ZFs) and transcription activator-like effectors (TALEs), researchers quickly expanded the CRISPR-Cas toolbox to include transcriptional activation, repression, and epigenetic modification. In this review, we highlight advances that extend the CRISPR-Cas toolkit for transcriptional and epigenetic regulation, as well as best practice guidelines for these tools, and a perspective on future applications.

Purpose: To establish if impression cytology combined with histochemical and immunocytochemical staining can be used to assess epithelium of the human upper lid margin. Methods: Following an initial eye examination of 40 healthy subjects (19 soft contact lens wearers and 21 non-contact lens wearers, aged 18–57 years), lid margin staining was assessed with lissamine green using slit lamp biomicroscopy and graded (grade 0–3). Impression cytology of the upper lid margin of both eyes was collected, fixed and stained with periodic acid Schiff (PAS) and haematoxylin for cell morphology analysis (Nelson grade) or for immunocytochemistry (keratinization-related proteins: filaggrin, transglutaminase1 (TGase1) and cytokeratin 1/10). Results: In 57% of all subjects, grade 0 lissamine green staining showed a thin line (the Marx line), just posterior to the meibomian gland ducts. Grade 2 or 3 lissamine green staining was observed in 17% of all subjects. There was no difference between contact lens and non-contact lens wearers for lid margin staining or Nelson grade (p = 0.4, Fisher’s exact test). PAS/haematoxylin staining and immunocytochemistry showed transition in epithelial cell morphology, with marginal conjunctival epithelium, mucocutaneous junction and squamous epithelium, adjacent to meibomian gland ducts. This transition in epithelium was associated with differential expression of keratinization-related proteins (filaggrin, cytokeratin 1/10 and TGase1). Conclusion: Lid margin epithelium can be successfully sampled using impression cytology and further characterized using histochemistry and immunocytochemistry staining techniques. This approach can be applied to assess lid margin changes in conditions such as dry eye and meibomian gland dysfunction.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver. © 2011 American Chemical Society.

MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice.Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. In this work, we developed a three-way electrochemical sensor for detection and quantitation of ultra-low amounts of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modes based on hybridization (H-SENS), p19 protein binding (P-SENS) and protein displacement (D-SENS). The combined HPD sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30\muL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 \muM. The HPD senor is made on a commercially-available gold nanoparticles-modified electrode and suitable for analysing multiple miRNAs on a single electrode. This three-way sensor exhibits high selectivity and specificity and was successfully used for sequential analysis of miR-32 and miR-122 on one electrode. Furthermore, the P-SENS was employed for profiling of three endogenous microRNAs (miR-21, miR-32 and miR-122) in human blood serum., This paper has been withdrawn by the author due to errors

Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes proceed with rate constants up to 3.38 ± 0.31 M -1 s -1 in CD 3CN, or 59 times faster than the analogous reaction of azides. This highly specific modular labeling strategy can be applied for direct labeling of proteins and for live cell imaging of cancer cells. © The Royal Society of Chemistry 2011.

MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19's utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1 fmol and a linear dynamic range from 1 pmol to 1 fmol. © 2011 Published by Elsevier Inc. All rights reserved.

Here we report that the phenanthridine derivative covalently linked to a fluorescein moiety (FLEth) can act as a fluorescence based probe for duplex short interfering RNA (siRNA) and that this probe can also be used to report on protein-RNA interactions. A fluorescence resonance energy transfer (FRET) signal that is observed at 600 nm occurs when FLEth is complexed with siRNA. At least 2 molecules of FLEth can bind to 21 nt duplex siRNA, and the dissociation constants for these interactions are reported. We find that FLEth can also report on the interaction of siRNAs with the Carnation Italian ringspot viral suppressor of RNA silencing p19. FLEth does not bind to the siRNA-p19 complex nor can p19 bind to the siRNA-FLEth complex; rather FLEth can report on the fraction of siRNA that is unbound. FLEth can also bind siRNA in delivery systems such as liposomes. Once the siRNA reaches the interior of Huh 7.5 cells, FLEth dissociates from the siRNA and is found in the nucleoli suggesting that FLEth cannot bind to siRNAs that are associated with the RNA silencing machinery.

Employees in nail salons, largely Vietnamese immigrant women in Boston, are exposed to a range of volatile organic chemicals from the products used in salons, including solvents, glues and polishes. Some of these chemicals have the potential to cause short and long-term adverse health effects. Only limited research has been performed on assessing occupational exposures. This project aimed to characterize total volatile organic compound (TVOC) and PM2.5 concentrations in nail salons as a function of ventilation, building characteristics, customer and employee occupancy, and type of services being performed. Students conducted sampling in 21 salons in Boston, MA from September to December, 2011. Study visits included: indoor environmental quality measurements (TVOCs, PM2.5 and carbon dioxide), site observations, and an interview. CO2 levels in 15 of 21 salons exceeded 800 ppm, suggesting that these salons may have insufficient ventilation. Higher TVOC and PM2.5 levels were found in salons with less ventilation (as estimated using CO2 concentrations). Contrary to our a priori hypothesis, average levels of TVOCs, CO2 and PM2.5 were consistent throughout salons, indicating that exposures may not be restricted to areas in the salon where work is being performed (e.g., at the manicure table). Higher TVOC concentrations were observed when tasks were being performed, yet were not dependent upon the number of tasks being performed. Improving ventilation conditions in salons to meet minimum outdoor air delivery requirements can reduce exposures to TVOCs.