P5396CRISPR/Cas9 mediated miR-29b editing restores muscle atrophy and exercise capacity in mice

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Li ◽  
L J Wang ◽  
F Wang ◽  
H F Tang ◽  
R Chen ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Genome Editing ◽  
Exercise Capacity ◽  
Muscle Atrophy ◽  
Intramuscular Administration ◽  
Loss Of Function ◽  
Guide Rna ◽  
Induced Apoptosis

Abstract Background Muscle atrophy is the loss of skeletal muscle mass and strength in response to diversity catabolic stimuli, such as heart failure. At present, no effective treatment except exercise is validated on reducing multiple muscle atrophy clinically. We have recently reported that microRNA-29b (miR-29b) promotes multiple types of muscle atrophy. Purpose The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system can efficiently introduce loss-of-function mutations into the endogenous miR-29b in vivo and as a potential therapy by treating muscle atrophy. Methods We used lentivirus to express CRISPR-associated 9 and a CRISPR guide RNA targeting miR-29b. Mutagenesis rate of miR-29b and off-target mutagenesis were detected by T7 Endonuclease I (T7EI) Assay. The expression level of miR-29b were measured in vitro and vivo after administration of the virus by using qRT-PCR. After intramuscular administration of the virus, the angiotensin II (AngII), immobilization and denervation-induced muscle atrophy were performed. Then muscle function was assessed in exercise capacity, the appearance and weight of muscle, the size of the muscle fibers, molecular and cellular detection. Results Here, we report that CRISPR/Cas9 mediated genome editing through intramuscular administration efficiently targeting the biogenesis processing sites in pre-miR-29b. No off-target mutagenesis was detected in 10 selected sites. This CRISPR-based treatment resulted in decreased miR-29b levels specifically. In vivo, this CRISPR-based treatment could ameliorate the muscle atrophy induced by angiotensin II (AngII), immobilization and denervation via activation of PI3K-AKT-mTOR signaling pathway and protect against AngII-induced apoptosis in mice. Moreover, the exercise capacity is also significantly enhanced. Conclusion Our work establishes CRISPR/Cas9 based gene targeting on miRNA as a potential durable therapy for treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo. Acknowledgement/Funding The grants from National Natural Science Foundation of China (81722008, 91639101 and 81570362 to JJ Xiao)

scholarly journals SLC25A21 Suppresses Cell Growth in Bladder Cancer Via The Reactive Oxygen Species-Mediated Mitochondrion-Dependent Apoptosis Pathway

2021 ◽  
Author(s):  
Yong Wang ◽  
Jiawen Gao ◽  
Shasha Hu ◽  
Weiting Zeng ◽  
Hongjun Yang ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Treatment Strategies ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Apoptosis Pathway ◽  
Normal Tissues ◽  
Induced Apoptosis ◽  
New Diagnosis

Abstract Background: Bladder cancer (BCa) is a commonly diagnosed malignancy worldwide that has poor survival depending on its intrinsic biologic aggressiveness and a peculiar radio- and chemoresistance features. Gaining a better understanding of tumorigenesis and developing new diagnosis and treatment strategies for BCa is important for improving BCa clinical outcome. SLC25 family member 21 (SLC25A21), a carrier transporting C5-C7 oxodicarboxylates, has been reported to contribute to oxoadipate acidemia. However, the potential role of SLC25A21 in cancer remains absolutely unknown. Methods: The expression levels of SLC25A21 in BCa and normal tissues were examined by real-time PCR and immunohistochemistry. Gain-of- and loss-of-function experiments were performed to detect the biological functions of SLC25A21 in vitro and in vivo by CCK-8 assay, plate colony formation assay, cell migration, invasion assay and experimental animal models. The subcellular distribution of substrate mediated by SLC25A21, mitochondrial membrane potential and ROS production were assessed to explore the potential mechanism of SLC25A21 in BCa.Results: We found that the expression of SLC25A21 was downregulated in BCa tissues compared to normal tissues. A significant positive correlation between decreased SLC25A21 expression and poor prognosis was observed in BCa patients. Overexpression of SLC25A21 significantly inhibited cell proliferation, migration and invasion and induced apoptosis in vitro. Moreover, the enhanced SLC25A21 expression significantly suppressed tumor growth in a xenograft mouse model. Furthermore, we revealed that SLC25A21 suppressed BCa growth by inducing the efflux of mitochondrial α-KG to the cytosol, decreasing to against oxidative stress, and activating the ROS-mediated mitochondrion-dependent apoptosis pathway. Conclusions: Our findings provide the first link between SLC25A21 expression and BCa and demonstrate that SLC25A21 acts as a crucial suppressor in BCa progression, which may help to provide new targets for BCa intervention.

scholarly journals Loss of Fas-signaling in pro-fibrotic fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis

2020 ◽  
Author(s):  
Elizabeth F. Redente ◽  
Sangeeta Chakraborty ◽  
Satria Sajuthi ◽  
Bart P. Black ◽  
Benjamin L. Edelman ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Lung Fibroblast ◽  
Lung Fibroblasts ◽  
Normal Lung ◽  
Loss Of Function ◽  
Fas Signaling ◽  
Distal Lung ◽  
Induced Apoptosis

ABSTRACTIdiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic disease of the distal lung alveoli that culminates in respiratory failure and reduced lifespan. Unlike normal lung repair in response to injury, IPF is associated with the accumulation and persistence of fibroblasts and myofibroblasts and continued production of collagen and other extracellular matrix (ECM) components. Prior in vitro studies have led to the hypothesis that the development of resistance to Fas-induced apoptosis by lung fibroblasts and myofibroblasts contibributes to their accumulation in the distal lung tissues of IPF patients. Here, we test this hypothesis in vivo in the resolving model of bleomycin-induced pulmonary fibrosis in mice. Using genetic loss-of-function approaches to inhibit Fas signaling in fibroblasts, novel flow cytometry strategies to quantify lung fibroblast subsets and transcriptional profiling of lung fibroblasts by bulk and single cell RNA-sequencing, we show that Fas is necessary for lung fibroblast apoptosis during homeostatic resolution of bleomycin-induced pulmonary fibrosis in vivo. Furthermore, we show that loss of Fas signaling leads to the persistence and continued pro-fibrotic functions of lung fibroblasts. Our studies provide novel insights into the mechanisms that contribute to fibroblast survival, persistence and continued ECM deposition in the context of IPF and how failure to undergo Fas-induced apoptosis prevents fibrosis resolution.

scholarly journals Disruption of Src function potentiates Chk1-inhibitor–induced apoptosis in human multiple myeloma cells in vitro and in vivo

Blood ◽  
2011 ◽  
Vol 117 (6) ◽  
pp. 1947-1957 ◽  
Author(s):  
Yun Dai ◽  
Shuang Chen ◽  
Rena Shah ◽  
Xin-Yan Pei ◽  
Li Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Xenograft Model ◽  
Mitogen Activated Protein Kinase ◽  
Loss Of Function ◽  
Compensatory Response ◽  
Human Multiple Myeloma ◽  
Chk1 Inhibitor ◽  
Induced Apoptosis

Abstract Ras/MEK/ERK pathway activation represents an important compensatory response of human multiple myeloma (MM) cells to checkpoint kinase 1 (Chk1) inhibitors. To investigate the functional roles of Src in this event and potential therapeutic significance, interactions between Src and Chk1 inhibitors (eg, UCN-01 or Chk1i) were examined in vitro and in vivo. The dual Src/Abl inhibitors BMS354825 and SKI-606 blocked Chk1-inhibitor–induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, markedly increasing apoptosis in association with BimEL up-regulation, p34cdc2 activation, and DNA damage in MM cell lines and primary CD138+ MM samples. Loss-of-function Src mutants (K297R, K296R/Y528F) or shRNA knock-down of Src prevented the ERK1/2 activation induced by Chk1 inhibitors and increased apoptosis. Conversely, constitutively active Ras or mitogen-activated protein kinase/ERK kinase 1 (MEK1) significantly diminished the ability of Src inhibitors to potentiate Chk1-inhibitor lethality. Moreover, Src/Chk1-inhibitor cotreatment attenuated MM-cell production of vascular endothelial growth factor and other angiogenic factors (eg, ANG [angiogenin], TIMP1/2 [tissue inhibitor of metalloproteinases 1/2], and RANTES [regulated on activation normal T-cell expressed and secreted]), and inhibited in vitro angiogenesis. Finally, coadministration of BMS354825 and UCN-01 suppressed human MM tumor growth in a murine xenograft model, increased apoptosis, and diminished angiogenesis. These findings suggest that Src kinase is required for Chk1-inhibitor–mediated Ras → ERK1/2 signaling activation, and that disruption of this event sharply potentiates the anti-MM activity of Chk1 inhi-bitors in vitro and in vivo.

scholarly journals A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos

Development ◽  
2021 ◽  
pp. dev.193565
Author(s):  
Shashank Gandhi ◽  
Yuwei Li ◽  
Weiyi Tang ◽  
Jens B. Christensen ◽  
Hugo A. Urrutia ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Function ◽  
Chick Embryos ◽  
Fluorescent Marker ◽  
Guide Rna ◽  
Single Plasmid ◽  
Lineage Analysis

An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian (RIA) retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly-engineered constructs and viruses by perturbing β-catenin in vitro and Sox10, Pax6, and Pax7 in the neural crest, retina, neural tube and segmental plate in vivo, respectively. Together, this approach enables knocking out genes of interest in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

scholarly journals Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3

2021 ◽  
Vol 118 (10) ◽  
pp. e2020401118
Author(s):  
Min Qiu ◽  
Zachary Glass ◽  
Jinjin Chen ◽  
Mary Haas ◽  
Xin Jin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Messenger Rna ◽  
Lipoprotein Metabolism ◽  
Density Lipoprotein ◽  
Blood Lipid ◽  
Loss Of Function ◽  
Guide Rna ◽  
Lipid Nanoparticle ◽  
Delivery Platform

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9–based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

scholarly journals Propofol Protects Rat Cardiomyocytes from Anthracycline-Induced Apoptosis by Regulating MicroRNA-181a In Vitro and In Vivo

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Hongwei Zhao ◽  
Xiaobei Zhang ◽  
Ying Zheng ◽  
Yuan Li ◽  
Xiaokun Wang ◽  
...  
Keyword(s):  
Myocardial Cell ◽  
Heart Tissue ◽  
Tunel Staining ◽  
Apoptotic Cells ◽  
Loss Of Function ◽  
Rat Cardiomyocytes ◽  
Direct Target Gene ◽  
Induced Apoptosis

We aimed to evaluate the cardioprotective effect and mechanism of propofol in anthracycline-induced cardiomyocyte apoptosis. We selected the rat myocardial cell line, H9c2, and primary cardiomyocytes for in vitro study. The cardiomyocytes were treated with vehicle, Adriamycin® (ADM), propofol, or a combination of ADM and propofol. The proportion of apoptotic cells and the expression of miR-181a were detected by flow cytometry and real-time PCR, respectively. Luciferase assays were performed to explore the direct target gene of miR-181a. In vivo assay, rats were randomly divided into different treatment groups. The apoptosis index was determined by TUNEL staining, and the expression of miR-181a and STAT3 in heart tissue was detected. The antiproliferative effect of ADM alone was significantly greater than that of ADM plus propofol. A significantly greater decrease in the proportion of apoptotic cells and in miR-181a expression was observed in the combination treatment group compared with that in the ADM groups in vitro and in vivo. The loss-of-function of miR-181a in H9c2 of ADM treatment resulted in increased Bcl-2 and decreased Bax. MiR-181a suppressed Bcl-2 expression through direct targeting of the Bcl-2 transcript. Propofol reduced anthracycline-induced apoptosis in cardiomyocytes via targeting miR-181a/Bcl-2, and a negative correlation between miR-181a and Bcl-2 was observed.

scholarly journals Long non-coding RNA SLC2A1-AS1 induced by GLI3 promotes aerobic glycolysis and progression in esophageal squamous cell carcinoma by sponging miR-378a-3p to enhance Glut1 expression

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Hongtao Liu ◽  
Qing Zhang ◽  
Yinsen Song ◽  
Yibin Hao ◽  
Yunxia Cui ◽  
...  
Keyword(s):  
Aerobic Glycolysis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Metabolic Remodeling ◽  
Glut1 Expression ◽  
Regulatory Feedback Loop ◽  
Induced Apoptosis

Abstract Background Emerging evidence demonstrates that lncRNAs play pivotal roles in tumor energy metabolism; however, the detailed mechanisms of lncRNAs in the regulation of tumor glycolysis remain largely unknown. Methods The expression of SLC2A1-AS1 was investigated by TCGA, GEO dataset and qRT-PCR. The binding of GLI3 to SLC2A1-AS1 promoter was detected by Luciferase Reporter Assay System and Ago2-RIP assay. FISH was performed to determine the localization of SLC2A1-AS1 in ESCC cells. Double Luciferase Report assay was used to investigate the interaction of miR-378a-3p with SLC2A1-AS1 and Glut1. Gain-of-function and Loss-of-function assay were performed to dissect the function of SLC2A1-AS1/miR-378a-3p/Glut1 axis in ESCC progression in vitro and in vivo. Results We identified a novel lncRNA SLC2A1-AS1 in ESCC. SLC2A1-AS1 was frequently overexpressed in ESCC tissues and cells, and its overexpression was associated with TNM stage, lymph node metastasis and poor prognosis of ESCC patients. Importantly, GLI3 and SLC2A1-AS1 formed a regulatory feedback loop in ESCC cells. SLC2A1-AS1 promoted cell growth in vitro and in vivo, migration and invasion, and suppressed apoptosis, leading to EMT progression and increased glycolysis in ESCC cells. SLC2A1-AS1 functioned as ceRNA for sponging miR-378a-3p, resulting in Glut1 overexpression in ESCC cells. MiR-378a-3p inhibited cell proliferation and invasion as well as induced apoptosis, resulting in reduced glycolysis, which was partly reversed by SLC2A1-AS1 or Glut1 overexpression in ESCC cells. Conclusion SLC2A1-AS1 plays important roles in ESCC development and progression by regulating glycolysis, and SLC2A1-AS1/miR-378a-3p/Glut1 regulatory axis may be a novel therapeutic target in terms of metabolic remodeling of ESCC patients.

scholarly journals A Conserved Myc Protein Domain, MBIV, Regulates DNA Binding, Apoptosis, Transformation, and G2 Arrest

2006 ◽  
Vol 26 (11) ◽  
pp. 4226-4239 ◽  
Author(s):  
Victoria H. Cowling ◽  
Sanjay Chandriani ◽  
Michael L. Whitfield ◽  
Michael D. Cole
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Binding Activity ◽  
Protein Domain ◽  
Loss Of Function ◽  
Dna Binding Activity ◽  
Induced Apoptosis

ABSTRACT The myc family of oncogenes is well conserved throughout evolution. Here we present the characterization of a domain conserved in c-, N-, and L-Myc from fish to humans, N-Myc317-337, designated Myc box IV (MBIV). A deletion of this domain leads to a defect in Myc-induced apoptosis and in some transformation assays but not in cell proliferation. Unlike other Myc mutants, MycΔMBIV is not a simple loss-of-function mutant because it is hyperactive for G2 arrest in primary cells. Microarray analysis of genes regulated by N-MycΔMBIV reveals that it is weakened for transactivation and repression but not nearly as defective as N-MycΔMBII. Although the mutated region is not part of the previously defined DNA binding domain, we find that N-MycΔMBIV has a significantly lower affinity for DNA than the wild-type protein in vitro. Furthermore, chromatin immunoprecipitation shows reduced binding of N-MycΔMBIV to some target genes in vivo, which correlates with the defect in transactivation. Thus, this conserved domain has an unexpected role in Myc DNA binding activity. These data also provide a novel separation of Myc functions linked to the modulation of DNA binding activity.

scholarly journals A total synthetic approach to CRISPR/Cas9 genome editing and homology directed repair

2018 ◽  
Author(s):  
Sara E. DiNapoli ◽  
Raul Martinez-McFaline ◽  
Caitlin K. Gribbin ◽  
Paul Wrighton ◽  
Courtney A. Balgobin ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Cell Types ◽  
Specific Cell ◽  
Synthetic Approach ◽  
Loss Of Function ◽  
Dna Templates ◽  
Homology Directed Repair

ABSTRACTCRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 142
Author(s):  
K. M. Whitworth ◽  
S. L. Murphy ◽  
J. A. Benne ◽  
L. D. Spate ◽  
E. Walters ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Genome Editing ◽  
Genetically Modified ◽  
Donor Cell ◽  
T7 Promoter ◽  
Guide Rna ◽  
Rag2 Gene

Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models. This study was funded by the National Institutes of Health via U42 OD011140.

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