Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.
When attempting to integrate single-molecule fluorescence microscopy with microfabricated devices such as microfluidic channels, fabrication constraints may prevent using traditional coverslips. Instead, the fabricated devices may require imaging through material with a different thickness or index of refraction. Altering either can easily reduce the quality of the image formation (measured by the Strehl ratio) by a factor of 2 or more, reducing the signal-to-noise ratio accordingly. In such cases, successful detection of single-molecule fluorescence may prove difficult or impossible. Here we provide software to calculate the effect of non-design materials upon the Strehl ratio or ensquared energy and explore the impact of common materials used in microfabrication.
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 CRISPRbased 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.
Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response to nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. We observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.
Majumder, Erica L.W.; Wolf, Benjamin M.; Liu, Haijun; Berg, R.H.; Timlin, Jerilyn A.; Chen, Min; Blankenship, Robert E.
Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. In this study, cyanobacteria containing chlorophyll f, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris, were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl f was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.
Herein, we describe a novel multifunctional metal–organic framework (MOF) materials platform that displays both porosity and tunable emission properties as a function of the metal identity (Eu, Nd, and tuned compositions of Nd/Yb). Their emission collectively spans the deep red to near-infrared (NIR) spectral region (~614–1350 nm), which is highly relevant for in vivo bioimaging. These new materials meet important prerequisites as relevant to biological processes: they are minimally toxic to living cells and retain structural integrity in water and phosphate-buffered saline. To assess their viability as optical bioimaging agents, we successfully synthesized the nanoscale Eu analog as a proof-of-concept system in this series. In vitro studies show that it is cell-permeable in individual RAW 264.7 mouse macrophage and HeLa human cervical cancer tissue culture cells. The efficient discrimination between the Eu emission and cell autofluorescence was achieved with hyperspectral confocal fluorescence microscopy, used here for the first time to characterize MOF materials. Importantly, this is the first report that documents the long-term conservation of the intrinsic emission in live cells of a fluorophore-based MOF to date (up to 48 h). As a result this finding, in conjunction with the materials’ very low toxicity, validates the biocompatibility in these systems and qualifies them as promising for use in long-term tracking and biodistribution studies.
Photosystem I (PSI) is the dominant photosystem in cyanobacteria and it plays a pivotal role in cyanobacterial metabolism. Despite its biological importance, the native organization of PSI in cyanobacterial thylakoid membranes is poorly understood. Here, we use atomic force microscopy (AFM) to show that ordered, extensive macromolecular arrays of PSI complexes are present in thylakoids from Thermosynechococcus elongatus, Synechococcus sp PCC 7002, and Synechocystis sp PCC 6803. Hyperspectral confocal fluorescence microscopy and three-dimensional structured illumination microscopy of Synechocystis sp PCC 6803 cells visualize PSI domains within the context of the complete thylakoid system. Crystallographic and AFM data were used to build a structural model of a membrane landscape comprising 96 PSI trimers and 27,648 chlorophyll a molecules. Rather than facilitating intertrimer energy transfer, the close associations between PSI primarily maximize packing efficiency; short-range interactions with Complex I and cytochrome b6f are excluded from these regions of the membrane, so PSI turnover is sustained by long-distance diffusion of the electron donors at the membrane surface. Elsewhere, PSI-photosystem II contact zones provide sites for docking phycobilisomes and the formation of megacomplexes. PSI-enriched domains in cyanobacteria might foreshadow the partitioning of PSI into stromal lamellae in plants, similarly sustained by long-distance diffusion of electron carriers.
Cosmic ray spikes are especially problematic for hyperspectral imaging because of the large number of spikes often present and their negative effects upon subsequent chemometric analysis. Fortunately, while the large number of spectra acquired in a hyperspectral imaging data set increases the probability and number of cosmic spikes observed, the multitude of spectra can also aid in the effective recognition and removal of the cosmic spikes. Zhang and Ben-Amotz were perhaps the first to leverage the additional spatial dimension of hyperspectral data matrices (DM). They integrated principal component analysis (PCA) into the upper bound spectrum method (UBS), resulting in a hybrid method (UBS-DM) for hyperspectral images. Here, we expand upon their use of PCA, recognizing that principal components primarily present in only a few pixels most likely correspond to cosmic spikes. Eliminating the contribution of those principal components in those pixels improves the cosmic spike removal. Both simulated and experimental hyperspectral Raman image data sets are used to test the newly developed UBS-DM-hyperspectral (UBS-DM-HS) method which extends the UBS-DM method by leveraging characteristics of hyperspectral data sets. A comparison is provided between the performance of the UBS-DM-HS method and other methods suitable for despiking hyperspectral images, evaluating both their ability to remove cosmic ray spikes and the extent to which they introduce spectral bias.
Fungi in the Candida genus are the most common fungal pathogens. They not only cause high morbidity and mortality but can also cost billions of dollars in healthcare. To alleviate this burden, early and accurate identification of Candida species is necessary. However, standard identification procedures can take days and have a large false negative error. The method described in this study takes advantage of hyperspectral confocal fluorescence microscopy, which enables the capability to quickly and accurately identify and characterize the unique autofluorescence spectra from different Candida species with up to 84% accuracy when grown in conditions that closely mimic physiological conditions.
Analyses of hyperspectral images with multivariate curve resolution (MCR) can be enhanced with the use of automated data preprocessing and improved MCR methods described and demonstrated in this chapter. These new approaches serve to greatly reduce or eliminate the need for user input and to increase the success, sensitivity, and accuracy of hyperspectral image analyses. We have pioneered the use of a dark spectral region, which is a general approach that can be introduced into any hyperspectral imaging system, to automatically remove offsets, structured noise, and uninformative pixels from the raw spectral images without the requirement of user input. The dark spectral region can also be used to minimize mixing between MCR-estimated spectral components to further improve the accuracy of the final pure spectra and their corresponding concentration images. The success of these improved preprocessing and MCR analysis methods is demonstrated with both realistically simulated images and experimentally measured spectral images.
Toomey, Matthew B.; Collins, Aaron M.; Frederiksen, Rikard; Cornwall, M.C.; Timlin, Jerilyn A.; Corbo, Joseph C.
The brilliantly coloured cone oil droplets of the avian retina function as long-pass cut-off filters that tune the spectral sensitivity of the photoreceptors and are hypothesized to enhance colour discrimination and improve colour constancy. Although it has long been known that these droplets are pigmented with carotenoids, their precise composition has remained uncertain owing to the technical challenges of measuring these very small, dense and highly refractile optical organelles. In this study, we integrated results from high-performance liquid chromatography, hyperspectral microscopy and microspectrophotometry to obtain a comprehensive understanding of oil droplet carotenoid pigmentation in the chicken (Gallus gallus). We find that each of the four carotenoid-containing droplet types consists of a complex mixture of carotenoids, with a single predominant carotenoid determining the wavelength of the spectral filtering cut-off. Consistent with previous reports, we find that the predominant carotenoid type in the oil droplets of long-wavelength-sensitive, medium-wavelength-sensitive and short-wavelength-sensitive type 2 cones are astaxanthin, zeaxanthin and galloxanthin, respectively. In addition, the oil droplet of the principal member of the double cone contains a mixture of galloxanthin and two hydroxycarotenoids (lutein and zeaxanthin). Short-wavelength-absorbing apocarotenoids are present in all of the droplet types, providing filtering of light in a region of the spectrum where filtering by hydroxy- and ketocarotenoids may be incomplete. Thus, birds rely on a complex palette of carotenoid pigmentswithin their cone oil droplets to achieve finely tuned spectral filtering.
Photosynthetic organisms use complex and regulated multichromophore assemblies, called lightharvesting (LH) antennas, to capture, concentrate and direct solar radiation to reaction centers that then carry out concomitant chemistry. Nature’s LH antennas are remarkable, operating with high efficiency in fluctuating environmental and photic conditions as well as being assembled with nanoscale precision thus, they often serve as inspiration in material design. The presented work was inspired by a natural LH antenna. We show that a diblock copolymer amphiphile enables the generation and integration of optically dense chromophore arrays, within a biomimetic polymer membrane. The entire construct is solution-processable, scalable and exhibits intra and inter-supramolecular energy transfer in a completely noncovalent design. This work demonstrates the potential of polymer membrane materials in generating spatial-energetic landscapes for photonic applications.
We assess the measurement of hyperspectral reflectance for the outdoor monitoring of green algae and cyanobacteria cultures with a multi-channel, fiber-coupled spectroradiometer. Reflectance data acquired over a four-week period are interpreted via numerical inversion of a reflectance model, in which the above-water reflectance is expressed as a quadratic function of the single backscattering albedo, dependent on the absorption and backscatter coefficients. The absorption coefficient is treated as the sum of component spectra consisting of the cultured species (green algae or cyanobacteria), dissolved organic matter, and water (including the temperature dependence of the water absorption spectrum). The backscatter coefficient is approximated as the scaled Hilbert transform of the culture absorption spectrum with a wavelength-independent vertical offset. Additional terms in the reflectance model account for the pigment fluorescence features and the water surface reflection of sunlight and skylight. For both the green algae and cyanobacteria, the wavelength-independent vertical offset of the backscatter coefficient is found to scale linearly with daily dry weight measurements, providing the capability for a non-sampling measurement of biomass in outdoor ponds. Other fitting parameters in the reflectance model are compared to auxiliary measurements and physics-based calculations. The magnitudes of the sunlight and skylight water-surface contributions derived from the reflectance model compare favorably with Fresnel reflectance calculations, while the reflectance-derived quantum efficiency of Chl-a fluorescence is found to be in agreement with literature values. To conlclude, the water temperature derived from the reflectance model exhibits excellent agreement with thermocouple measurements during the morning hours and highlights significantly elevated temperatures in the afternoon hours.
Microalgae have been identified as a promising renewable feedstock for production of lipids for feeds and fuels. Current methods for identifying algae strains and growth conditions that support high lipid production require a variety of fluorescent chemical indicators, such as Nile Red and more recently, Bodipy. Despite notable successes using these approaches, chemical indicators exhibit several drawbacks, including non-uniform staining, low lipid specificity, cellular toxicity, and variable permeability based on cell-type, limiting their applicability for high-throughput bioprospecting. In this work, we used in vivo hyperspectral confocal fluorescence microscopy of a variety of potential microalgae production strains (Nannochloropsis sp., Dunaliella salina, Neochloris oleoabundans, and Chlamydomonas reinhardtii) to identify a label-free method for localizing lipid bodies and quantifying the lipid yield on a single-cell basis. By analyzing endogenous fluorescence from chlorophyll and resonance Raman emission from lipid-solubilized carotenoids we deconvolved pure component emission spectra and generated diffraction limited projections of the lipid bodies and chloroplast organelles, respectively. Applying this imaging method to nutrient depletion time-courses from lab-scale and outdoor cultivation systems revealed an additional autofluorescence spectral component that became more prominent over time, and varied inversely with the chlorophyll intensity, indicative of physiological compromise of the algal cell. This signal could result in false-positives for conventional measurements of lipid accumulation (via spectral overlap with Nile Red), however, the additional spectral feature was found to be useful for classification of lipid enrichment and culture crash conditions in the outdoor cultivation system. Under nutrient deprivation, increases in the lipid fraction of the cellular volume of ~. 500% were observed, as well as a correlated decrease in the chloroplast fraction of the total cellular volume. The results suggest that a membrane recycling mechanism dominates for nutrient deprivation-based lipid accumulation in the microalgae tested.
Engineered nanomaterials (ENMs) are increasingly being used in commercial products, particularly in the biomedical, cosmetic, and clothing industries. For example, pants and shirts are routinely manufactured with silver nanoparticles to render them 'wrinkle-free.' Despite the growing applications, the associated environmental health and safety (EHS) impacts are completely unknown. The significance of this problem became pervasive within the general public when Prince Charles authored an article in 2004 warning of the potential social, ethical, health, and environmental issues connected to nanotechnology. The EHS concerns, however, continued to receive relatively little consideration from federal agencies as compared with large investments in basic nanoscience R&D. The mounting literature regarding the toxicology of ENMs (e.g., the ability of inhaled nanoparticles to cross the blood-brain barrier; Kwon et al., 2008, J. Occup. Health 50, 1) has spurred a recent realization within the NNI and other federal agencies that the EHS impacts related to nanotechnology must be addressed now. In our study we proposed to address critical aspects of this problem by developing primary correlations between nanoparticle properties and their effects on cell health and toxicity. A critical challenge embodied within this problem arises from the ability to synthesize nanoparticles with a wide array of physical properties (e.g., size, shape, composition, surface chemistry, etc.), which in turn creates an immense, multidimensional problem in assessing toxicological effects. In this work we first investigated varying sizes of quantum dots (Qdots) and their ability to cross cell membranes based on their aspect ratio utilizing hyperspectral confocal fluorescence microscopy. We then studied toxicity of epithelial cell lines that were exposed to different sized gold and silver nanoparticles using advanced imaging techniques, biochemical analyses, and optical and mass spectrometry methods. Finally we evaluated a new assay to measure transglutaminase (TG) activity; a potential marker for cell toxicity.