RESUMO
Seven pairs of undescribed monoterpenoid polyprenylated acylphloroglucinol enantiomers [(±)-hypermonanones A-G (1-7)], together with three known analogues, were identified from the whole plant of Hypericum monanthemum Hook. The structures of these compounds were determined by analyses of their UV, HRESIMS, 1D/2D NMR spectroscopic data, and NMR calculations. The absolute configurations of these compounds were assigned by ECD calculations after chiral HPLC separation. Diverse monoterpene moieties were fused at C-3/C-4 of the dearomatized acylphloroglucinol core, which led to 3,4-dihydro-2H-pyran-integrated angular or linear type 6/6/6 tricyclic skeletons in 1-7. Compounds (-)-2 and (+)-2 exhibited significant NO inhibitory activity against LPS induced RAW264.7 cells with the IC50 values of 7.07 ± 1.02 µM and 11.39 ± 0.24 µM, respectively.
Assuntos
Hypericum , Monoterpenos , Floroglucinol , Compostos Fitoquímicos , Hypericum/química , Camundongos , Estrutura Molecular , Monoterpenos/isolamento & purificação , Monoterpenos/farmacologia , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Floroglucinol/química , Células RAW 264.7 , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Animais , Óxido Nítrico/metabolismo , Estereoisomerismo , ChinaRESUMO
Garciyunnanones A-R (1-18), eighteen undescribed caged polycyclic polyprenylated acylphloroglucinols, two undescribed biogenetic congeners (19-20), and nineteen known analogues (21-39), were isolated from the stem barks of Garcinia yunnanensis Hu. All of these isolates are decorated with a C-5 lavandulyl substituent. Their structures and absolute configurations were confirmed by HRESIMS, 1D & 2D NMR spectroscopic analysis, quantum chemical calculations of electronic circular dichroism data, and single-crystal X-ray diffraction analysis. The X-ray crystallographic data of ten isolated caged compounds ascertained the absolute configuration of C-23 in the lavandulyl as S. The cytotoxicity on three cancer cell lines and the anti-nonalcoholic steatohepatitis activity of the isolates were tested. In a free fatty acid-induced L02 cell model, compounds 33 and 39 decreased intracellular lipid accumulation significantly.
Assuntos
Antineoplásicos Fitogênicos , Garcinia , Floroglucinol , Garcinia/química , Humanos , Floroglucinol/química , Floroglucinol/farmacologia , Floroglucinol/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral , Modelos Moleculares , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Casca de Planta/químicaRESUMO
Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.
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Diferenciação Celular , Ventrículos Laterais , Fator Inibidor de Leucemia , Organoides , Células-Tronco Pluripotentes , Humanos , Organoides/metabolismo , Organoides/citologia , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Fator de Transcrição STAT3/metabolismo , Neuroglia/metabolismo , Neuroglia/citologia , Transdução de SinaisRESUMO
Therapeutic strategies are the hot-spot issues in treating patients with advanced oral squamous cell carcinoma (OSCC). Mounting studies have proved that triggering ferroptosis is one of the promising targets for OSCC management. In this study, we performed a first attempt to collect the current evidence on the proposed roles of ferroptosis in OSCC through a comprehensive review. Based on clinical data from the relevant studies within this topic, we found that ferroptosis-associated tumor microenvironment, ferroptosis-related genes (FRGs), and ferroptosis-related lncRNAs exhibited a potent prognostic value for OSCC patients. Mechanistically, experimental data revealed that the proliferation and tumorigenesis of OSCC might be associated with the inhibition of cellular ferroptosis through the activation of glutathione peroxidase 4 (GPX4) and adipocyte enhancer-binding protein 1 (AEBP1), suppression of glutathione (GSH) and Period 1 (PER1) expression, and modulation of specific non-coding RNAs (i.e., miR-520d-5p, miR-34c-3p, and miR-125b-5p) and their targeted proteins. Several specific interventions (i.e., Quisinostat, Carnosic acid, hyperbaric oxygen, melatonin, aqueous-soluble sporoderm-removed G. lucidum spore powder, and disulfiram/copper complex) were found to dramatically induce ferroptosis cell death of OSCC via multiple mechanisms. This review highlighted the pivotal role of ferroptosis in the pathogenesis and prognosis of OSCC. Future anticancer therapeutic strategies targeting ferroptosis and its associated molecules might provide a new insight for OSCC treatment.
Assuntos
Carcinoma de Células Escamosas , Ferroptose , Neoplasias Bucais , Ferroptose/genética , Humanos , Neoplasias Bucais/patologia , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Prognóstico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Microambiente TumoralRESUMO
Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.
Assuntos
Transporte Axonal , Cinesinas , Animais , Axônios , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Mitocôndrias/metabolismoRESUMO
Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small deletions, or insertions at a specific genomic locus, a strategy that shows great promise for creating "Disease in a dish" models. To improve the effectiveness of prime editing in hPSCs, we systematically compared and combined the "inhibition of mismatch repair pathway and p53" on top of the "PEmax" to generate an all-in-one "PE-Plus" prime editor. We show that PE-Plus conducts the most efficient editing among the current PE tools in hPSCs. We further established an inducible prime editing platform in hPSCs by incorporating the all-in-one PE vector into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Therefore, our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.
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A meta-analysis was performed to investigate the efficacy of ultrapulse carbon dioxide dot matrix laser treatment for patients with facial scars. PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure, China Biomedical Literature Database, and Wanfang Database were systematically searched for randomised controlled trials (RCTs) investigating ultrapulse carbon dioxide dot matrix laser treatment for facial scars, and the search was conducted from the time of database inception to July 2023. The retrieved literature was screened independently by two researchers, and data extraction and quality assessments were performed. The meta-analysis was conducted using RevMan 5.4 software. Outcome metrics included overall treatment effectiveness, complication rate, and Echelle d'évaluation clinique des cicatrices d'acné (ECCA) scores. Seventeen RCTs comprising 3703 patients were included, with 1853 patients in the experimental group and 1850 in the control group. The results showed that the experimental group had significantly increased overall treatment efficacy rates (odds ratio [OR]: 3.84, 95% confidence interval [CI]: 3.02-4.90, p < 0.001), reduced complication rates (OR: 0.35, 95% CI: 0.27-0.44, p < 0.001), and improved ECCA scores (standardised mean difference: -1.79, 95% CI: -2.53 to -1.05, p < 0.001) compared with the control group. In conclusion, as the primary treatment modality for facial acne depression scars, ultrapulse carbon dioxide dot matrix laser can significantly increase the overall treatment efficacy rate and ECCA scores and reduce the incidence of complications; however, higher-quality studies are needed for further validation.
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Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1's regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1's mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1's O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.
RESUMO
An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Espectrina , Animais , Transporte Axonal , Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Espectrina/metabolismoRESUMO
Prime editing introduces single-nucleotide polymorphism changes, small deletions, or insertions at a specific genome site without double-stranded DNA breaks or the need for the donor template. Here, we present a protocol to design, conduct, and evaluate prime editing in human pluripotent stem cells. We describe steps for pegRNA and nicking sgRNA design and cloning, the prime editing tool electroporation, and the efficiency evaluation using Miseq. We elaborate the process of GBA (N370S) mutation induction and correction as an example. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes , Humanos , RNA Guia de Sistemas CRISPR-Cas , Mutação , Genoma , Edição de Genes/métodosRESUMO
Largemouth bass (Micropterus salmoides) is an important economic freshwater aquaculture fish originating from North America. However, the frequent outbreaks of Micropterus salmoides rhabdovirus (MSRV) have seriously limited the healthy development of Micropterus salmoides farming industry. In the present study, a strain of MSRV was isolated and identified from infected largemouth bass by PCR, transmission electron micrograph observation and genome sequences analysis, and tentatively named MSRV-HZ01 strain. Phylogenetic analyses showed that the MSRV-HZ01 presented the highest similarity to MSRV-2021, followed by MSRV-FJ985 and MSRV-YH01. The various tissues of juvenile largemouth bass exhibited significant pathological damage following MSRV-HZ01 immersion infection, and the mortality reached 90%. We also found that intestine was the key organ for MSRV to enter the fish body initially by dynamic analysis of viral infection, and the head kidney was the susceptible tissue of virus. Moreover, the MSRV was also transferred to the external mucosal tissue in later stage of viral infection to achieve horizontal transmission. In addition, the genes of IFN γ and IFN I-C were significantly up-regulated after MSRV infection to exert antiviral functions. The genes of cGAS and Sting might play an important role in the regulation of interferon expression. In conclusion, we investigated the virus infection dynamics and fish response following MSRV immersion infection, which would promote our understanding of the interaction between MSRV and largemouth bass under natural infection.
Assuntos
Bass , Doenças dos Peixes , Rhabdoviridae , Viroses , Animais , Bass/genética , Filogenia , ImersãoRESUMO
Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport.
RESUMO
Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.
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A 28 day feeding trial was conducted to investigate the growth performance, immune response and intestinal microbiota of laminarin (LAM) supplemented diets in juvenile largemouth bass (Micropterus salmoides). Four hundred and eighty fish (initial average weight: 0.72 ± 0.04 g) were randomly divided into four groups (40 fish per tank with three replicates in each group) Four diets were prepared with LAM supplementation at the doses of 0 (control), 5 g Kg-1 (LL), 10 g Kg-1 (ML) and 15 g Kg-1 (HL), respectively. No significant difference in the specific growth rate (SGR) and hepatosomatic index (HSI) was observed in fish among the four groups, or in the lipid and ash content of fish flesh. In addition, fish in the LL group exhibited much higher antioxidant capacity (p < 0.05), while the diets with the inclusion of 5 and 10 g Kg-1 LAM remarkably decreased the antioxidant capacity of fish (p > 0.05). Dietary LAM at the dose of 5 g Kg-1 inhibited the transcription of interleukin-1ß (il-1ß) and tumor necrosis factor-α (tnf-α), while promoting the expression of transforming growth factor-ß (tgf-ß) in fish intestine. Moreover, the beneficial intestinal bacteria Bacteroide, Comamonas and Mycoplasma abundance significantly increased in fish from the LL group, while the content of opportunistic pathogens Plesiomonas, Aeromonas and Brevinema in fish of the HL group was substantially higher than the control group. Overall, the appropriate dose of supplemented LAM in the diet was 5 g Kg-1, while an excessive supplementation of LAM in the diet led to microbial community instability in largemouth bass.
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Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Proteína Supressora de Tumor p53/genética , Citosina , Edição de Genes/métodos , Células-Tronco Pluripotentes/fisiologia , Sistemas CRISPR-CasRESUMO
Nasopharyngeal carcinoma (NPC) has high incidence in China and East and Southeast Asia. The study was performed to investigate the effect of microRNA3942-3p (miR-3942-3p) on the radiosensitivity of NPC. Compared with non-cancer tissue, NPC had significantly lower miR-3942-3p expression. X-irradiation (IR) reduced the expression of miR-3942-3p in a dose-dependent way in NPC cells. Down-regulation of miR-3942-3p using miR-3942-3p inhibitor resulted in significantly increased cell viability, decreased apoptosis of CNE1 cells. Bax decreased and Bcl2 increased after IR. The expression of BARD1, a cancer predisposing gene, was elevated in NPC tissue. It was confirmed to be a target of miR-3942-3p using luciferase reporter assay. Down-regulation of BARD1 using siRNA significantly reduced cell viability and significantly increased apoptosis both before and after IR. The same response was observed when miR-3942-3p mimics was used to transfect BARD1-overexpressing CNE1 cells, suggesting the up-regulation of miR-3942-3p could sensitize CNE1 cells to X-rays via BARD1. Our data demonstrate that up-regulation of miR-3942-3p could sensitize NPC to X-rays via a downstream target BARD1, offering potential new strategies for radiotherapy of NPC.
Assuntos
MicroRNAs , Neoplasias Nasofaríngeas , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/radioterapia , Tolerância a Radiação/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In Caenorhabditis elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 activates the conserved Sarm1/TIR-1-ASK1/NSY-1-p38 MAPK pathway and eventually the transcription factor CEBP-1 to protect against degeneration. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated, and uncover a surprising neuroprotective role for the Sarm1-p38 MAPK pathway in this context.
Within the cell are various compartments that carry out specific roles. This includes the mitochondria, which are responsible for generating the chemical energy that powers the cell. Some of the most power-hungry cells are nerve cells, which have long, slender projections called axons that relay signals from one part of the nervous system to another. If the mitochondria do not work properly, the axons break down which can lead to neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Previous studies showed that defects in how mitochondria are transported cause axons in the roundworm Caenorhabditis elegans to spontaneously deteriorate with age. These transparent worms are often used to study biological questions related to the nervous system. Here, Ding et al. have used this model organism to identify a molecular pathway that stops axons from degenerating. Random mutations were introduced into the genome of C. elegans that are unable to transport mitochondria in to their axons. Ding et al. then searched for mutant strains that still had intact axons despite this mitochondrial defect. This revealed that mutations that activate a protein called CaMKII stop axons from breaking down. Further experiments showed that CAMKII does this by switching on a series of signals, including the protein Sarm1, that eventually turn on another protein that suppresses degeneration. The protective role of Sarm1 is surprising given that this protein has been shown to promote axon degeneration after injury in flies and mammals. This suggests that the gene for Sarm1 as well as others likely play different roles depending on the context in which they are activated. These findings could help researchers identify new drug targets and strategies for treating neurodegenerative diseases caused by mitochondrial defects. However, further work is needed to see if this newly discovered pathway works the same way in other model systems, such as flies and mammals.
Assuntos
Proteínas do Domínio Armadillo , Proteínas de Caenorhabditis elegans , Animais , Proteínas do Domínio Armadillo/metabolismo , Axônios/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.