Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.

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Raman spectroscopic markers have been determined for fatigue-related microdamage in bovine bone. Microdamage was induced using a cyclic fatigue loading regime. After loading, the specimens were stained en-bloc with basic fuchsin to facilitate damage visualization and differentiate fatigue-induced damage from cracks generated during subsequent histological sectioning. Bone tissue specimens were examined by light microscopy and hyperspectral near-infrared Raman imaging microscopy. Three regions were defined-tissue with no visible damage, tissue with microcracks, and tissue with diffuse damage. Raman transects, lines of 150-200 Raman spectra, were used for initial tissue surveys. Exploratory factor analysis of the transect Raman spectra has identified spectroscopically distinct chemical microstructures of the bone specimens that correlate with damage. In selected regions of damage, full hyperspectral Raman images were obtained with 1.4-microm spatial resolution. In regions of undamaged tissue, the phosphate nu1 band is found at 957 cm(-1), as expected for the carbonated hydroxyapatic bone mineral. However, in regions of visible microdamage, an additional phosphate nu1 band is observed at 963 cm(-1) and interpreted as a more stoichiometric, less carbonated mineral species. Raman imaging confirms the qualitative relationship between the Raman spectral signature of bone mineral and the type of microdamage in bovine bone. Two tentative explanations for the presence of less carbonated phosphate in damaged regions are proposed.

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (Ϸ0.03-m 3 ) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the shortwavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.cyanobacteria ͉ photosynthetic pigments ͉ multivariate curve resolution C yanobacteria convert light energy to chemical energy by means of photosynthesis, using water as a source of electrons for CO 2 reduction and O 2 production. A key part of the photosynthesis process is light absorption (harvesting) by pigments, followed by excitation transfer to reaction center chlorophyll (Chl) a of photosystems (PS) II and I (1). These processes take place in thylakoid membranes that in cyanobacteria generally form an extensive internal membrane complex of several layers along the periphery of the cytoplasm, with thylakoids found less frequently toward the center of the cell (2).The pigments associated with the photosynthetic apparatus are bound to thylakoid proteins, modifying their spectral properties and providing a spatial distribution that aids in the efficiency of light harvesting and energy transfer to reaction center Chls. Pigments bound to integral membrane proteins in reaction center complexes in thylakoids of cyanobacteria include Chl a [Ϸ40 per PS II (3) and Ϸ100 per PS I (4)] and carotenoids; the latter act in photoprotection and 3 Chl quenchin...

We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.