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Deepthi Alapati, Edward E Morrisey
While our understanding of the genetics and pathology of congenital lung diseases such as surfactant protein deficiency, cystic fibrosis and alpha 1 antitrypsin deficiency is extensive, treatment options are lacking. Since the lung is a barrier organ in direct communication with the external environment, targeted delivery of gene corrective technologies to the respiratory system via intra-tracheal or intranasal routes is an attractive option for therapy. CRISPR/Cas9 gene editing technology is a promising approach to repair or inactivate disease causing mutations...
October 25, 2016: American Journal of Respiratory Cell and Molecular Biology
Elizabeth A Simonik, Ying Cai, Katherine N Kimmelshue, Dana M Brantley-Sieders, Holli A Loomans, Claudia D Andl, Grant M Westlake, Victoria M Youngblood, Jin Chen, Wendell G Yarbrough, Brandee T Brown, Lalitha Nagarajan, Stephen J Brandt
Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease...
2016: PloS One
Shilpa Nagaraju, Naomi Kathleen Davies, David Jeffrey Fraser Walker, Michael Köpke, Séan Dennis Simpson
BACKGROUND: Impactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts like Escherichia coli or yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential. RESULTS: In this study, we report scarless genome editing of an industrially used model acetogen, Clostridium autoethanogenum, using the CRISPR/Cas9 system...
2016: Biotechnology for Biofuels
Xing Liu, Xiaolian Cai, Bo Hu, Zhichao Mei, Dawei Zhang, Gang Ouyang, Jing Wang, Wei Zhang, Wuhan Xiao
FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor-α (HIF-α) degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown...
October 24, 2016: Journal of Biological Chemistry
Jihye Chung, Shunsuke Aburaya, Wataru Aoki, Mitsuyoshi Ueda
In very early stages of cancer development, one or a few cells expressing cancer-associated genes appear among a much larger number of surrounding normal cells. To analyze the molecular changes induced by this co-existence, we artificially prepared transformed cells with complete loss of tumor suppressor gene, SCRIB, among normal human embryonic kidney (HEK293T) cells. A cell strain with SCRIB-knockout was successfully constructed by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease system and co-cultured with normal cells...
October 21, 2016: Journal of Bioscience and Bioengineering
Kaihang Wang, Julius Fredens, Simon F Brunner, Samuel H Kim, Tiongsun Chia, Jason W Chin
Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection...
October 24, 2016: Nature
Wenshu Luo, Hidenobu Mizuno, Ryohei Iwata, Shingo Nakazawa, Kosuke Yasuda, Shigeyoshi Itohara, Takuji Iwasato
Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood...
October 24, 2016: Scientific Reports
Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology. Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA (sgRNA)-mediated Cas9 cutting of target DNA, and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair. Firstly, four sgRNAs were designed for Rev-erbβ gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pCMV-hCas9-U6-Rev-erbβ sgRNA1 and pCMV-hCas9-U6-Rev-erbβ sgRNA2...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Julien Muffat, Yun Li, Rudolf Jaenisch
In vitro differentiation of human pluripotent stem cells provides a systematic platform to investigate the physiological development and function of the human nervous system, as well as the etiology and consequence when these processes go awry. Recent development in three-dimensional (3D) organotypic culture systems allows modeling of the complex structure formation of the human CNS, and the intricate interactions between various resident neuronal and glial cell types. Combined with an ever-expanding genome editing and regulation toolkit such as CRISPR/Cas9, it is now a possibility to study human neurological disease in the relevant molecular, cellular and anatomical context...
October 18, 2016: Current Opinion in Cell Biology
Nina Xie, He Gong, Joshua A Suhl, Pankaj Chopra, Tao Wang, Stephen T Warren
Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies...
2016: PloS One
Glenn Yiu, Eric Tieu, Anthony T Nguyen, Brittany Wong, Zeljka Smit-McBride
Purpose: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. Methods: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene...
October 1, 2016: Investigative Ophthalmology & Visual Science
Li-Wa Shao, Rong Niu, Ying Liu
Neurons have a central role in the systemic coordination of mitochondrial unfolded protein response (UPR(mt)) and the cell non-autonomous modulation of longevity. However, the mechanism by which the nervous system senses mitochondrial stress and communicates to the distal tissues to induce UPR(mt) remains unclear. Here we employ the tissue-specific CRISPR-Cas9 approach to disrupt mitochondrial function only in the nervous system of Caenorhabditis elegans, and reveal a cell non-autonomous induction of UPR(mt) in peripheral cells...
October 21, 2016: Cell Research
Ludovic Enkler, Delphine Richer, Anthony L Marchand, Dominique Ferrandon, Fabrice Jossinet
Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C...
October 21, 2016: Scientific Reports
Ching-Tzu Yen, Meng-Ni Fan, Yung-Li Yang, Sheng-Chieh Chou, I-Shing Yu, Shu-Wha Lin
Hemophilia is the most well-known hereditary bleeding disorder, with an incidence of one in every 5000 to 30,000 males worldwide. The disease is treated by infusion of protein products on demand and as prophylaxis. Although these therapies have been very successful, some challenging and unresolved tasks remain, such as reducing bleeding rates, presence of target joints and/or established joint damage, eliminating the development of inhibitors, and increasing the success rate of immune-tolerance induction (ITI)...
2016: Thrombosis Journal
Fan Lin, Liang Dong, Weiming Wang, Yuchen Liu, Weiren Huang, Zhiming Cai
Optogenetic gene expression systems enable spatial-temporal modulation of gene transcription and cell behavior. Although applications in biomedicine are emerging, the utility of optogenetic gene switches remains elusive in cancer research due to the relative low gene activation efficiency. Here, we present an optimized CRISPR-Cas9-based light-inducible gene expression device that controls gene transcription in a dose-dependent manner. To prove the potential utility of this device, P53 was tested as a functional target in the bladder cancer cell models...
2016: International Journal of Biological Sciences
James West, W Warren Gill
Genome editing in large animals has tremendous practical applications, from more accurate models for medical research through improved animal welfare and production efficiency. Although genetic modification in large animals has a 30 year history, until recently technical issues limited its utility. The original methods - pronuclear injection and integrating viruses - were plagued with problems associated with low efficiency, silencing, poor regulation of gene expression, and variability associated with random integration...
June 2016: Journal of Equine Veterinary Science
Katsuya Sato, Masashi Kimura, Kazue Sugiyama, Masashi Nishikawa, Yukio Okano, Hitoshi Nagaoka, Takahiro Nagase, Yukio Kitade, Hiroshi Ueda
PLEKHG2/FLJ00018 is a Gβγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e., FHL1A, FHL1B and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2...
October 20, 2016: Journal of Biological Chemistry
Steven J Kleene, Nancy K Kleene
Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening monogenic renal disease. ADPKD results from mutations in either of two proteins: polycystin-1 (also known as PC1 or PKD1) or transient receptor potential cation channel, subfamily P, member 2 (TRPP2, also known as polycystin-2, PC2, or PKD2). Each of these proteins is expressed in the primary cilium that extends from many renal epithelial cells. Existing evidence suggests that the cilium can promote renal cystogenesis, while PC1 and TRPP2 counter this cystogenic effect...
October 19, 2016: American Journal of Physiology. Renal Physiology
Xiaolong Wang, Bei Cai, Jiankui Zhou, Haijing Zhu, Yiyuan Niu, Baohua Ma, Honghao Yu, Anmin Lei, Hailong Yan, Qiaoyan Shen, Lei Shi, Xiaoe Zhao, Jinlian Hua, Xingxu Huang, Lei Qu, Yulin Chen
Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced...
2016: PloS One
Kristin Nicole Harper
No abstract text is available yet for this article.
October 14, 2016: AIDS
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