Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.

The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.

Reversible suppression of cyclooxygenase 2 (COX-2) expression in vivo by inducible RNA interference.

Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3'untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.

Application of a rapid, simple, and accurate adenovirus-based method to compare PET reporter gene/PET reporter probe systems.

PURPOSE: This study aims to use a simple, quantitative method to compare the HSV1sr39TK/(18) F-FHBG PET reporter gene/PET reporter probe (PRG/PRP) system with PRGs derived from human nucleoside kinases. PROCEDURES: The same adenovirus vector is used to express alternative PRGs. Equal numbers of vectors are injected intravenously into mice. After PRP imaging, quantitative hepatic PET signals are normalized for transduction by measuring hepatic viral genomes. RESULTS: The same adenovirus vector was used to express equivalent amounts of HSV1sr39TK, mutant human thymidine kinase 2 (TK2-DM), and mutant human deoxycytidine kinase (dCK-A100VTM) in mouse liver. HSV1sr39TK expression was measured with (18) F-FHBG, TK2-DM and dCK-A100VTM with (18) F-L-FMAU. TK2-DM/(18) F-L-FMAU and HSV1sr39TK/(18) F-FHBG had equivalent sensitivities; dCK-A100VTM/(18) F-L-FMAU was twice as sensitive as HSV1sr39TK/(18) F-FHBG. CONCLUSIONS: The human PRG/PRP sensitivities are comparable and/or better than HSV1sr39TK/(18) F-FHBG. However, for clinical use, identification of the best PRP substrate for each enzyme, characterization of probe distribution, and consequences of overexpressing nucleoside kinases must be evaluated.

A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols.

Production of green chemicals and fuels using metabolically engineered organisms has been a promising alternative to petroleum-based production. Higher chain alcohols (C4-C8) are of interest because they can be used as chemical feedstock as well as fuels. Recently, the feasibility of n-hexanol synthesis using Escherichia coli has been demonstrated by extending the modified Clostridium CoA-dependent n-butanol synthesis pathway, thereby elongating carbon chain length via reactions in reversed ß-oxidation, (or ß-reduction). Here, we developed an anaerobic growth selection platform that allows selection or enrichment of enzymes for increased synthesis of C6 and C8 linear alcohols. Using this selection, we were able to improve the carbon flux towards the synthesis of C6 and C8 acyl-CoA intermediates. Replacement of the original enzyme Clostridium acetobutylicum Hbd with Ralstonia eutropha homologue PaaH1 increased production of n-hexanol by 10-fold. Further directed evolution by random mutagenesis of PaaH1 improved n-hexanol and n-octanol production. This anaerobic growth selection platform may be useful for selecting enzymes for production of long-chain alcohols and acids using this CoA-dependent pathway.

A method to rapidly and accurately compare the relative efficacies of non-invasive imaging reporter genes in a mouse model and its application to luciferase reporters.

PURPOSE: Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure and compare the luciferase reporter efficacies. PROCEDURES: Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring that the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. RESULTS: We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed greater than 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc and greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. CONCLUSIONS: Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET).

In vivo mouse bioluminescence tomography with radionuclide-based imaging validation.

INTRODUCTION: Bioluminescence imaging, especially planar bioluminescence imaging, has been extensively applied in in vivo preclinical biological research. Bioluminescence tomography (BLT) has the potential to provide more accurate imaging information due to its 3D reconstruction compared with its planar counterpart. METHODS: In this work, we introduce a positron emission tomography (PET) radionuclide imaging-based strategy to validate the BLT results. X-ray computed tomography, PET, spectrally resolved bioluminescence imaging, and surgical excision were performed on a tumor xenograft mouse model expressing a bioluminescent reporter gene. RESULTS: With different spectrally resolved measured data, the BLT reconstructions were acquired based on the third-order simplified spherical harmonics (SP3) approximation and the diffusion approximation (DA). The corresponding tomographic images were obtained for validation of bioluminescence source reconstruction. CONCLUSION: Our results show the strength of PET imaging compared with other validation methods for BLT and improved source localization accuracy based on the SP(3) approximation compared with the diffusion approximation.

Positron emission tomography imaging of DMBA/TPA mouse skin multi-step tumorigenesis.

Many tumor cells have elevated rates of glucose uptake that can be measured quantitatively, noninvasively and repeatedly by positron emission tomography (PET) with 2-deoxy-2-[(18)F]-fluoro-D-glucose ((18)F-FDG). Clinical imaging with (18)F-FDG PET has been used for detection and staging of primary and metastatic tumors. High-resolution microPET scanning and murine cancer models make it possible to analyze longitudinally glucose metabolism during the appearance, development and progression of individual experimental tumors. In this study, we used (18)F-FDG microPET and micro computerized tomography (microCT) to investigate glucose uptake in the DMBA/TPA chemically-induced multistage mouse skin carcinogenesis model. (18)F-FDG uptake is significantly higher in all papillomas than in surrounding skin. Elevated (18)F-FDG uptake is observed when tumors can be identified morphologically, but not before. Although (18)F-FDG uptake is high in all fully invasive, malignant skin squamous cell carcinomas, uptake in papillomas and microinvasive malignant squamous cell carcinomas is variable and does not exhibit any correlation with tumor stage.

Spectrally resolved bioluminescence tomography with the third-order simplified spherical harmonics approximation.

Bioluminescence imaging has been extensively applied to in vivo small animal imaging. Quantitative three-dimensional bioluminescent source information obtained by using bioluminescence tomography can directly and much more accurately reflect biological changes as opposed to planar bioluminescence imaging. Preliminary simulated and experimental reconstruction results demonstrate the feasibility and promise of bioluminescence tomography. However, the use of multiple approximations, particularly the diffusion approximation theory, affects the quality of in vivo small animal-based image reconstructions. In the development of new reconstruction algorithms, high-order approximation models of the radiative transfer equation and spectrally resolved data introduce new challenges to the reconstruction algorithm and speed. In this paper, an SP(3)-based (the third-order simplified spherical harmonics approximation) spectrally resolved reconstruction algorithm is proposed. The simple linear relationship between the unknown source distribution and the spectrally resolved data is established in this algorithm. A parallel version of this algorithm is realized, making BLT reconstruction feasible for the whole body of small animals especially for fine spatial domain discretization. In simulation validations, the proposed algorithm shows improved reconstruction quality compared with diffusion approximation-based methods when high absorption, superficial sources and detection modes are considered. In addition, comparisons between fine and coarse mesh-based BLT reconstructions show the effects of numerical errors in reconstruction image quality. Finally, BLT reconstructions using in vivo mouse experiments further demonstrate the potential and effectiveness of the SP(3)-based reconstruction algorithm.

Experimental bioluminescence tomography with fully parallel radiative-transfer-based reconstruction framework.

Bioluminescence imaging is a very sensitive imaging modality, used in preclinical molecular imaging. However, in its planar projection form, it is non-quantitative and has poor spatial resolution. In contrast, bioluminescence tomography (BLT) promises to provide three dimensional quantitative source information. Currently, nearly all BLT reconstruction algorithms in use employ the diffusion approximation theory to determine light propagation in tissues. In this process, several approximations and assumptions that are made severely affect the reconstruction quality of BLT. It is therefore necessary to develop novel reconstruction methods using high-order approximation models to the radiative transfer equation (RTE) as well as more complex geometries for the whole-body of small animals. However, these methodologies introduce significant challenges not only in terms of reconstruction speed but also for the overall reconstruction strategy. In this paper, a novel fully-parallel reconstruction framework is proposed which uses a simplified spherical harmonics approximation (SPN). Using this framework, a simple linear relationship between the unknown source distribution and the surface measured photon density can be established. The distributed storage and parallel operations of the finite element-based matrix make SPN-based spectrally resolved reconstruction feasible at the small animal whole body level. Performance optimization of the major steps of the framework remarkably improves reconstruction speed. Experimental reconstructions with mouse-shaped phantoms and real mice show the effectiveness and potential of this framework. This work constitutes an important advance towards developing more precise BLT reconstruction algorithms that utilize high-order approximations, particularly second-order self-adjoint forms to the RTE for in vivo small animal experiments.

The influence of innate and pre-existing immunity on adenovirus therapy.

Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre-existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed. Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre-existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad-immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre-existing immunity.

The MAPK pathway is required for depolarization-induced "promiscuous" immediate-early gene expression but not for depolarization-restricted immediate-early gene expression in neurons.

Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD-IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The "promiscuous" IEGs Egr1 and c-fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl-induced Egr1 and c-fos expression. In contrast, MEK1/2 inhibition has no effect on KCl-induced expression of the known IPD-IEGs in PC12 cells. Additional "candidate" IDP-IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD-IEGs, as with the known IPD-IEGs, are also induced by a MAP kinase- independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD-IEGs. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl-induced IPD-IEG mRNA accumulation, suggesting that the IPD-IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA- and CaMKII-dependent pathways.

The COX-2 inhibitor parecoxib produces neuroprotective effects in MPTP-lesioned rats.

The present study investigated the effects of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib (Bextratrade mark) in the prevention of motor and cognitive impairments observed in rats after an intranigral infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a model of the early phase of Parkinson's disease. The treatment with parecoxib (10 mg/kg) administered prior to the surgery and daily (2 mg/kg) for the subsequent 21 days, prevented the MPTP-treated rats from presenting decreased locomotor and exploratory behavior, increased immobility, and impairment while performing the cued version of the Morris water maze. Furthermore, parecoxib treatment also significantly prevented the reduction of tyrosine hydroxylase protein expression in the substantia nigra (7, 14 and 21 days after surgery), and in the striatum (14 and 21 days after surgery) as immunodetected by western blotting. These results strongly suggest that parecoxib exerts a neuroprotective effect on motor, tyrosine hydroxylase expression, and cognitive functions as it prevents their impairments within the confines of this animal model of the early phase of Parkinson's disease.

MAPKAP kinase-2 is a primary response gene induced by depolarization in PC12 cells and in brain.

Using a combination of targeted differential display for induced protein kinases and differential library screening, we identified mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2), as a primary response gene whose transcription is stimulated by membrane depolarization and by forskolin in rat PC12 pheochromocytoma cells. MAPKAPK3 was neither induced nor repressed by similar treatments. The increase in MAPKAPK2 mRNA is preceded by an increase in a MAPKAPK2 intron-containing RNA precursor, indicating that the increase in message is due at least in part to increased transcription. The open reading frame of full-length rat MAPKAPK2 cDNA is 99% identical to mouse MAPKAPK2 and 92% identical to human MAPKAPK2. The human MAPKAPK2 predicted protein contains 14 additional amino acids in the proline-rich N-terminal domain, when compared to murine and rat MAPKAPK2 predicted proteins. The MAPKAPK2 form found in PC12 cells corresponds to variant 2 in the human; this ortholog carries a nuclear translocation signal near its C-terminus. MAPKAPK2 message is also induced in the dentate gyrus, CA1, and CA3 of the rat hippocampus between 2-4 hr after the onset of kainic acid-induced seizures.

Synaptotagmin IV overexpression inhibits depolarization-induced exocytosis in PC12 cells.

Depolarization-induced vesicle exocytosis is a complex mechanism involving a number of proteins. In this process, synaptotagmins work as members of the Ca(2+)-sensing system that triggers the fusion of the synaptic vesicle with the plasma membrane. Synaptotagmin IV (SytIV), an immediate-early gene induced by depolarization in PC12 pheochromocytoma cells and in the hippocampus, has been suggested to work as a negative regulator of neurotransmitter release. Unlike other synaptotagmins, SytIV has an evolutionarily conserved substitution of an aspartate to a serine in the Ca(2+) coordination site of its C2A domain, preventing SytIV from binding anionic lipids in a Ca(2+)-dependent fashion. We used the secretion of human growth hormone (hGH) as a reporter system with which to examine the effects of overexpressing SytIV and other depolarization-induced immediate-early genes (the protein kinases KID-1, SIK, and PIM-1 and the transcription factors rTLE3 and Nurr1) on depolarization-induced vesicle exocytosis in PC12 cells. SytIV overexpression resulted in decreased depolarization-induced hGH release. However, conversion of the unique serine in SytIV to an aspartate eliminated this inhibitory activity. In addition, rTLE3 overexpression produced only a modest increase in spontaneous vesicle exocytosis, whereas KID-1, SIK, PIM-1, and Nurr1 overexpression had no effect on depolarization-induced exocytosis.

Expression of depolarization-induced immediate early gene proteins in PC12 cells.

Immediate early genes induced by depolarization are thought to be important in mediating neuronal functional plasticity. We previously identified a group of immediate early genes that are preferentially induced by depolarization and forskolin but not by nerve growth factor or epidermal growth factor in PC12 pheochromocytoma cells. These depolarization-induced genes include synaptotagmin 4; the protein kinases KID-1, PIM-1, and SIK; an orphan transcription factor, Nurr-1; and a transcription corepressor, rTLE-3. All these genes are also induced in the hippocampus in response to kainic-acid induced depolarization. To characterize further the unique functions of these genes in plasticity, we used recombinant proteins to generate and purify antibodies against KID-1 and SIK proteins. Immunoblotting experiments were performed to examine the induced expression of the KID-1 and SIK proteins in PC12 cells. PIM-1 and Nurr-1 protein expression was also examined following stimulation, using commercially available antibodies. There is an increase in synthesis, in PC12 cells, of these four IEG proteins after KCl plus forskolin treatment. Nurr-1 protein peaks between 2 and 4 hr and decreases by 6 hr after the treatment. PIM-1 and KID-1 proteins rise by 1 hr, peak between 2 and 4 hr, and return to their basal levels at 6 hr. SIK protein increases significantly at 2 hr after treatment, peaks between 4 and 6 hr, and returns to the basal level at 8 hr. Immunofluorescence studies demonstrate distinct distribution patterns of each of these depolarization-induced IEG proteins in PC12 cells.