scholarly journals Single adeno-associated virus-based multiplexed CRISPR-Cas9 system to nullify core components of the mammalian molecular clock

2020 ◽  
Author(s):  
Boil Kim ◽  
Jihoon Kim ◽  
Minjeong Chun ◽  
Inah Park ◽  
Mijung Choi ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Molecular Clock ◽  
Viral Vectors ◽  
Clock Genes ◽  
Ex Vivo ◽  
Physiological Role ◽  
Guide Rnas ◽  
Multiple Genes

ABSTRACTThe mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) containing Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1). TTFL robustness is endowed by genetic complementation between these components; therefore, multiple genes must be knocked out to physiologically investigate the molecular clock, which requires extensive research resources. To facilitate molecular clock disruption, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for Pers, Crys, or Bmal1. First, we designed single guide RNAs (sgRNAs) targeting individual clock genes using an in silico approach and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and expressed in viral vectors. CSAC efficiency was demonstrated by decreased protein expression in vitro and ablated molecular oscillation ex vivo. We also measured locomotor activity and body temperature in Cas9-expressing mice injected with CSAC at the suprachiasmatic nucleus. Circadian rhythm disruption was observed under free-running conditions, indicating that CSAC can efficiently and robustly disrupt molecular circadian clock. Thus, CSAC is a simple and powerful tool for investigating the physiological role of the molecular clock in vivo.

scholarly journals Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Boil Kim ◽  
Jihoon Kim ◽  
Minjeong Chun ◽  
Inah Park ◽  
Damhyeon Kwak ◽  
...  
Keyword(s):  
Molecular Clock ◽  
Clock Genes ◽  
Ex Vivo ◽  
Molecular Clocks ◽  
Genetic Redundancy ◽  
Efficient Manner ◽  
Knock Out ◽  
Temperature Rhythms

AbstractThe mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.

scholarly journals Cell type resolved co-expression networks of core clock genes in brain development

2021 ◽  
Author(s):  
Surbhi Sharma ◽  
Asgar Hussain Ansari ◽  
Soundhar Ramasamy
Keyword(s):  
Circadian Clock ◽  
Single Cell ◽  
Radial Glia ◽  
Clock Genes ◽  
Neuronal Cell ◽  
Cell Types ◽  
Developing Brain ◽  
Knock Out

AbstractThe circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimer’s and Parkinson’s. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.

scholarly journals Cancer clocks in tumourigenesis: the p53 pathway and beyond

2021 ◽  
Vol 28 (4) ◽  
pp. R95-R110
Author(s):  
Ewan M Stephenson ◽  
Laura E J Usselmann ◽  
Vinay Tergaonkar ◽  
David M Virshup ◽  
Robert Dallmann
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Cancer Biology ◽  
Cell Cycle Progression ◽  
Clock Genes ◽  
Human Cancer ◽  
Epidemiologic Studies ◽  
Current Evidence

Circadian rhythms regulate a vast array of physiological and cellular processes, as well as the hormonal milieu, to keep our cells synchronised to the light–darkness cycle. Epidemiologic studies have implicated circadian disruption in the development of breast and other cancers, and numerous clock genes are dysregulated in human tumours. Here we review the evidence that circadian rhythms, when altered at the molecular level, influence cancer growth. We also note some common pitfalls in circadian-cancer research and how they might be avoided to maximise comparable results and minimise misleading data. Studies of circadian gene mutant mice, and human cancer models in vitro and in vivo, demonstrate that clock genes can impact tumourigenesis. Clock genes influence important cancer-related pathways, ranging from p53-mediated apoptosis to cell cycle progression. Confusingly, clock dysfunction can be both pro- or anti-tumourigenic in a model and cell type-specific manner. Due to this duality, there is no canonical mechanism for clock interaction with tumourigenic pathways. To understand the role of the circadian clock in patients’ tumours requires analysis of the molecular clock status compared to healthy tissue. Novel mathematical approaches are under development, but this remains largely aspirational, and is hampered by a lack of temporal information in publicly available datasets. Current evidence broadly supports the notion that the circadian clock is important for cancer biology. More work is necessary to develop an overarching model of this connection. Future studies would do well to analyse the clock network in addition to alterations in single clock genes.

scholarly journals A Circadian Clock Entrained by Melatonin Is Ticking in the Rat Fetal Adrenal

Endocrinology ◽  
2011 ◽  
Vol 152 (5) ◽  
pp. 1891-1900 ◽  
Author(s):  
C. Torres-Farfan ◽  
N. Mendez ◽  
L. Abarzua-Catalan ◽  
N. Vilches ◽  
G. J. Valenzuela ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Circadian Clock ◽  
Clock Genes ◽  
Regulatory Protein ◽  
Fetal Life ◽  
Early Growth Response ◽  
Circadian Expression ◽  
Steroidogenic Acute Regulatory

The adrenal gland in the adult is a peripheral circadian clock involved in the coordination of energy intake and expenditure, required for adaptation to the external environment. During fetal life, a peripheral circadian clock is present in the nonhuman primate adrenal gland. Whether this extends to the fetal adrenal gland like the rat is unknown. Here we explored in vivo and in vitro whether the rat fetal adrenal is a peripheral circadian clock entrained by melatonin. We measured the 24-h changes in adrenal content of corticosterone and in the expression of clock genes Per-2 and Bmal-1 and of steroidogenic acute regulatory protein (StAR), Mt1 melatonin receptor, and early growth response protein 1 (Egr-1) expression. In culture, we explored whether oscillatory expression of these genes persisted during 48 h and the effect of a 4-h melatonin pulse on their expression. In vivo, the rat fetal adrenal gland showed circadian expression of Bmal-1 and Per-2 in antiphase (acrophases at 2200 and 1300 h, respectively) as well as of Mt1 and Egr-1. This was accompanied by circadian rhythms of corticosterone content and of StAR expression both peaking at 0600 h. The 24-h oscillatory expression of Bmal-1, Per-2, StAR, Mt1, and Egr-1 persisted during 48 h in culture; however, the antiphase between Per-2 and Bmal-1 was lost. The pulse of melatonin shifted the acrophases of all the genes studied and restored the antiphase between Per-2 and Bmal-1. Thus, in the rat, the fetal adrenal is a strong peripheral clock potentially amenable to regulation by maternal melatonin.

In Vivo Marking of Rhesus Monkey Lymphocytes by Adeno-Associated Viral Vectors: Direct Comparison With Retroviral Vectors

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2263-2270 ◽  
Author(s):  
Yutaka Hanazono ◽  
Kevin E. Brown ◽  
Atsushi Handa ◽  
Mark E. Metzger ◽  
Dominik Heim ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Dna Sequences ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Retroviral Vectors ◽  
Peripheral Blood Lymphocytes ◽  
Transduction Efficiency ◽  
Monkey Model

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated β-galactosidase gene (β-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.

scholarly journals Effects of propofol on mammalian central and peripheral circadian clocks

2019 ◽  
Author(s):  
Hongni Tian ◽  
Ziqing Yu ◽  
Xiwen Zhu ◽  
Yuanjing Chen ◽  
Qian Ren ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Clock Genes ◽  
Ex Vivo ◽  
Hypoxia Inducible Factor ◽  
Bzip Transcription Factor ◽  
General Anesthetic ◽  
Hypoxia Inducible Factor 1 ◽  
Mammalian Liver ◽  
Group D

Abstract Background Circadian rhythm has a significant correlation with the occurrence and development of many diseases. Studies have shown that the anesthetic agent propofol can alter the rhythms of body temperature and activity in rats. Methods U2OS cells and ex vivo liver tissue were treated with different concentration of propofol, followed by recording the oscillation of the circadian clock. And two month-old mice were exposed to propofol (10mg/kg and 20mg/kg) or vehicle, detecting the expression of the clock genes. Results The results showed that propofol reduced the amplitude and lengthened the period of Per2 oscillation. Treatment with 10 mg/kg propofol significantly increased the expression of D-box binding PAR BZIP transcription factor (Dbp) and clock circadian regulator (Clock) in the liver. Treatment with 20 mg/kg propofol significantly decreased expression of cryptochrome circadian regulator 1 (Cry1), Dbp, nuclear receptor subfamily 1 group D member 1 (Nr1d1) and Clock and significantly increased the hypoxia signaling pathway genes hypoxia inducible factor 1 subunit alpha (Hif-1α), Egl-9 family hypoxia inducible factors Egln1, Egln2 and Egln3 in the hypothalamus. Conclusion The above results indicate that the general anesthetic propofol can change the circadian clock of ex vivo and in vivo mammalian liver tissues.

scholarly journals Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors

2006 ◽  
Vol 80 (3) ◽  
pp. 1487-1496 ◽  
Author(s):  
Anne Keriel ◽  
Céline René ◽  
Chad Galer ◽  
Joseph Zabner ◽  
Eric J. Kremer
Keyword(s):  
Gene Transfer ◽  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Transduction Efficiency

ABSTRACT A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

scholarly journals The Core-Clock Gene NR1D1 Impacts Cell Motility In Vitro and Invasiveness in a Zebrafish Xenograft Colon Cancer Model

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 853 ◽  
Author(s):  
Alireza Basti ◽  
Rita Fior ◽  
Müge Yalҫin ◽  
Vanda Póvoa ◽  
Rosario Astaburuaga ◽  
...  
Keyword(s):  
Cell Migration ◽  
Circadian Clock ◽  
Cell Motility ◽  
Clock Genes ◽  
Xenograft Model ◽  
Migration And Invasion ◽  
Altered Expression ◽  
The Core

Malfunctions of circadian clock trigger abnormal cellular processes and influence tumorigenesis. Using an in vitro and in vivo xenograft model, we show that circadian clock disruption via the downregulation of the core-clock genes BMAL1, PER2, and NR1D1 impacts the circadian phenotype of MYC, WEE1, and TP53, and affects proliferation, apoptosis, and cell migration. In particular, both our in vitro and in vivo results suggest an impairment of cell motility and a reduction in micrometastasis formation upon knockdown of NR1D1, accompanied by altered expression levels of SNAI1 and CD44. Interestingly we show that differential proliferation and reduced tumour growth in vivo may be due to the additional influence of the host-clock and/or to the 3D tumour architecture. Our results raise new questions concerning host–tumour interaction and show that core-clock genes are involved in key cancer properties, including the regulation of cell migration and invasion by NR1D1 in zebrafish xenografts.

scholarly journals Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection

2013 ◽  
Vol 455 (2) ◽  
pp. 133-147 ◽  
Author(s):  
Michael Freeley ◽  
Aideen Long
Keyword(s):  
T Cells ◽  
Gene Silencing ◽  
Inflammatory Disease ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Sirna Delivery ◽  
Attractive Potential ◽  
Therapeutic Benefits

The specificity of RNAi and its ability to silence ‘undruggable’ targets has made inhibition of gene expression in T-cells with siRNAs an attractive potential therapeutic strategy for the treatment of inflammatory disease, cancer and infection. However, delivery of siRNAs into primary T-cells represents a major hurdle to their use as potential therapeutic agents. Recent advances in siRNA delivery through the use of electroporation/nucleofection, viral vectors, peptides/proteins, nanoparticles, aptamers and other agents have now enabled efficient gene silencing in primary T-cells both in vitro and in vivo. Overcoming such barriers in siRNA delivery offers exciting new prospects for directly targeting T-cells systemically with siRNAs, or adoptively transferring T-cells back into patients following ex vivo manipulation with siRNAs. In the present review, we outline the challenges in delivering siRNAs into primary T-cells and discuss the mechanism and therapeutic opportunities of each delivery method. We emphasize studies that have exploited RNAi-mediated gene silencing in T-cells for the treatment of inflammatory disease, cancer and infection using mouse models. We also discuss the potential therapeutic benefits of manipulating T-cells using siRNAs for the treatment of human diseases.

scholarly journals Recent Advances in CRISPR/Cas9 Delivery Strategies

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 839 ◽  
Author(s):  
Bon Ham Yip
Keyword(s):  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
Ex Vivo ◽  
Extracellular Vesicle ◽  
Delivery Methods ◽  
Delivery Strategies ◽  
In Vivo Delivery

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.

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