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Irene Vorontsova, Ines Gehring, James E Hall, Thomas F Schilling
Purpose: To investigate the roles of Aquaporin 0a (Aqp0a) and Aqp0b in zebrafish lens development and transparency. Methods: CRISPR/Cas9 gene editing was used to generate loss-of-function deletions in zebrafish aqp0a and/or aqp0b. Wild type (WT), single mutant, and double mutant lenses were analyzed from embryonic to adult stages. Lens transparency, morphology, and growth were assessed. Immunohistochemistry was used to map protein localization as well as to assess tissue organization and distribution of cell nuclei...
June 1, 2018: Investigative Ophthalmology & Visual Science
Emily A Stout, Rosemary Sanozky-Dawes, Yong Jun Goh, Alexandra B Crawley, Todd R Klaenhammer, Rodolphe Barrangou
Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system...
July 19, 2018: Microbiology
Katelyn Dunigan, Qian Li, Rui Li, Morgan L Locy, Stephanie B Wall, Trent E Tipple
Thioredoxin reductase-1 (TXNRD1) inhibition effectively activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and attenuates lung injury in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD) models. Upon TXNRD1 inhibition, heme oxygenase-1 (HO-1) is disproportionally increased compared with Nrf2 target NADPH quinone oxidoreductase-1 (NQO1). HO-1 has been investigated as a potential therapeutic target in both ARDS and BPD. TXNRD1 is predominantly expressed in airway epithelial cells; however, the mechanism of HO-1 induction by TXNRD1 inhibitors is unknown...
July 19, 2018: American Journal of Physiology. Lung Cellular and Molecular Physiology
Qiudi Deng, Zisheng Chen, Lei Shi, Huafeng Lin
The CRISPR/Cas9 system has been developed as a powerful tool for targeted gene editing. As a result of technical enhancements in recent years, this technology has become the method of choice for efficiently modifying targeted HIV-1 genome efficiently as part of HIV therapy. CRISPR can be modified to target specific sequences that Cas9 then cuts. In this article, we outline the development of the CRISPR/Cas9 system. We also show how this technology can be used for the prevention and treatment of HIV-1 infection...
July 19, 2018: Reviews in Medical Virology
Priya Dharmalingam, Hari Krishna R Rachamalla, Brijesh Lohchania, Bhanuprasad Bandlamudi, Saravanabhavan Thangavel, Mohankumar K Murugesan, Rajkumar Banerjee, Arabinda Chaudhuri, Chandrashekhar Voshavar, Srujan Marepally
Cationic lipid-guided nucleic acid delivery holds great promise in gene therapy and genome-editing applications for treating genetic diseases. However, the major challenge lies in achieving therapeutically relevant efficiencies. Prior findings, including our own, demonstrated that asymmetry in the hydrophobic core of cationic lipids imparted superior transfection efficiencies. To this end, we have developed a lipid nanocarrier system with an asymmetric hydrophobic core ( PS-Lips ) derived from a mixture of fatty acids of food-grade palmstearin and compared its efficiency with symmetric palmitic acid-based nanocarrier system ( P-Lip )...
November 30, 2017: ACS Omega
Hangjun Sun, Lili Shen, Yuanxia Qin, Xiaowei Liu, Kaiqiang Hao, Ying Li, Jie Wang, Jinguang Yang, Fenglong Wang
Chloride channel (CLC) proteins are important anion transporters conserved in organisms ranging from bacteria and yeast to plants and animals. According to sequence comparison, some plant CLCs are predicted to function as Cl- /H+ antiporters, but not Cl- channels. However, no direct evidence was provided to verify the role of these plant CLCs in regulating the pH of the intracellular compartment. We identified tobacco CLC-Nt1 interacting with the Potato virus Y (PVY) 6K2 protein. To investigate its physiological function, homologous genes of CLC-Nt1 in Nicotiana benthamiana were knocked out using the CRISPR/Cas9 system...
July 19, 2018: New Phytologist
Harveer Dev, Ting-Wei Will Chiang, Chloe Lescale, Inge de Krijger, Alistair G Martin, Domenic Pilger, Julia Coates, Matylda Sczaniecka-Clift, Wenming Wei, Matthias Ostermaier, Mareike Herzog, Jonathan Lam, Abigail Shea, Mukerrem Demir, Qian Wu, Fengtang Yang, Beiyuan Fu, Zhongwu Lai, Gabriel Balmus, Rimma Belotserkovskaya, Violeta Serra, Mark J O'Connor, Alejandra Bruna, Petra Beli, Luca Pellegrini, Carlos Caldas, Ludovic Deriano, Jacqueline J L Jacobs, Yaron Galanty, Stephen P Jackson
BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region...
July 18, 2018: Nature Cell Biology
Mrinal K Sarkar, Grace A Hile, Lam C Tsoi, Xianying Xing, Jianhua Liu, Yun Liang, Celine C Berthier, William R Swindell, Matthew T Patrick, Shuai Shao, Pei-Suen Tsou, Ranjitha Uppala, Maria A Beamer, Anshika Srivastava, Stephanie L Bielas, Paul W Harms, Spiro Getsios, James T Elder, John J Voorhees, Johann E Gudjonsson, J Michelle Kahlenberg
OBJECTIVE: Skin inflammation and photosensitivity are common in patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE), yet little is known about the mechanisms that regulate these traits. Here we investigate the role of interferon kappa (IFN-κ) in regulation of type I interferon (IFN) and photosensitive responses and examine its dysregulation in lupus skin. METHODS: mRNA expression of type I IFN genes was analysed from microarray data of CLE lesions and healthy control skin...
July 18, 2018: Annals of the Rheumatic Diseases
Xin Chen, Lina Kozhaya, Cihan Tastan, Lindsey Placek, Mikail Dogan, Meghan Horne, Rebecca Abblett, Ece Karhan, Martin Vaeth, Stefan Feske, Derya Unutmaz
Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes ( CD4 , CD45 , CD95 ) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency...
July 18, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
Javier T Granados-Riveron, Guillermo Aquino-Jarquin
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) system has been rapidly harnessed to perform various genomic engineering tasks. Recently, it has been demonstrated that a novel RNA-targeting CRISPR effector protein, called Cas13, binds and cleaves RNA rather than DNA substrates analogously to the eukaryotic RNA interference system. The known Cas13a-Cas13d effectors are able to efficiently cleave complementary target single-stranded RNAs, which represent a potentially safer alternative to deoxyribonuclease Cas9, because it induces loss-of-function phenotypes without genomic loss of the targeted gene...
July 18, 2018: Cancer Research
Hai Long Liu, Yang Shen, Yang Gao, Ling Zhou, Xiao Song Han, Chang Zhi Zhao, Gao Juan Yang, Yi Long Chen, Hui Yang, Sheng Song Xie
CRISPR/Cas technology enables efficient and specific editing the genome. Since different bacterial sources or artificially modified Cas9, as well as Cpf1 and other nucleases, recognize different PAMs (protospacer adjacent motifs), different gene editing nucleases may use different types of sgRNAs (small guide RNA). MicroRNAs (miRNAs) are a class of regulatory small non-coding RNAs. To determine whether specific targets for sgRNAs in miRNA precursor exit, the abundance and specificity of 11 different types of sgRNA targeting 28 645 miRNA precursors were analyzed in the present study using the CRISPR-offinder, a bioinformatics software developed in our own laboratory...
July 20, 2018: Yi Chuan, Hereditas
Michael A Gomez, Z Daniel Lin, Theodore Moll, Raj Deepika Chauhan, Luke Hayden, Kelley Renninger, Getu Beyene, Nigel J Taylor, J Carrington, B Staskawicz, R Bart
Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms...
July 18, 2018: Plant Biotechnology Journal
Roghayyeh Baghban, Safar Farajnia, Younes Ghasemi, Mojtaba Mortazavi, Nosratollah Zarghami, Naser Samadi
BACKGROUND: Although Pichia pastoris is an outstanding host among conventional expression systems for production of recombinant proteins, a new interest have been emerged to this system due to the inherent advantages and new developments in this expression host. The potential for secretory and soluble expression of heterologous glycoproteins in P. pastoris proposed this system as a candidate for the production of complex eukaryotic proteins. METHODS: Several new developments have been occurred in different areas related to P...
July 17, 2018: Current Pharmaceutical Biotechnology
Masakazu Yoshida, Etsuko Yokota, Tetsushi Sakuma, Tomoki Yamatsuji, Nagio Takigawa, Toshikazu Ushijima, Takashi Yamamoto, Takuya Fukazawa, Yoshio Naomoto
TP63 encodes TAp63, which is functionally similar to the tumor suppressor TP53 , and ΔNp63, which lacks the transcription-activating domain of TAp63 and appears potently oncogenic in squamous cell carcinomas (SCCs). In this study, we developed an integrated CRISPR interference (CRISPRi) system to selectively suppress ΔNp63 (CRISPRiΔNp63). We engineered this CRISPRi using tandemized guide RNA expression cassettes that targeted the 50 to 100 bp downstream of the transcription start site of ΔNp63 in combination with inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain...
June 26, 2018: Oncotarget
Ryosuke Hashimoto, Risa Ueta, Chihiro Abe, Yuriko Osakabe, Keishi Osakabe
Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various Cas9 expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express Cas9 ...
2018: Frontiers in Plant Science
Caroline Poyntner, Ursula Mirastschijski, Katja Sterflinger, Hakim Tafer
The black yeast Exophiala dermatitidis is a polyextremophilic human pathogen, especially known for growing in man-made extreme environments. Reported diseases caused by this fungus range from benign cutaneous to systemic infections with 40% fatality rate. While the number of cases steadily increases in both immunocompromised and immunocompetent people, detailed knowledge about infection mechanisms, virulence factors and host response are scarce. To understand the impact of the putative virulence factor melanin on the infection, we generated a polyketide synthase ( PKS1 ) mutant using CRISPR/Cas9 resulting in a melanin deficient strain...
2018: Frontiers in Microbiology
Carles Cornet, Vincenzo Di Donato, Javier Terriente
The use of zebrafish larvae in basic and applied research has grown exponentially during the last 20 years. The reasons for this success lay in its specific experimental advantages: on the one hand, the small size, the large number of progeny and the fast life cycle greatly facilitate large-scale approaches while maintaining 3Rs amenability; on the other hand, high genetic and physiological homology with humans and ease of genetic manipulation make zebrafish larvae a highly robust model for understanding human disease...
2018: Frontiers in Pharmacology
Yongmoon Jeon, You Hee Choi, Yunsu Jang, Jihyeon Yu, Jiyoung Goo, Gyejun Lee, You Kyeong Jeong, Seung Hwan Lee, In-San Kim, Jin-Soo Kim, Cherlhyun Jeong, Sanghwa Lee, Sangsu Bae
Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand...
July 17, 2018: Nature Communications
Danielle T Avery, Alisa Kane, Tina Nguyen, Anthony Lau, Akira Nguyen, Helen Lenthall, Kathryn Payne, Wei Shi, Henry Brigden, Elise French, Julia Bier, Jana R Hermes, David Zahra, William A Sewell, Danyal Butt, Michael Elliott, Kaan Boztug, Isabelle Meyts, Sharon Choo, Peter Hsu, Melanie Wong, Lucinda J Berglund, Paul Gray, Michael O'Sullivan, Theresa Cole, Steven M Holland, Cindy S Ma, Christoph Burkhart, Lynn M Corcoran, Tri Giang Phan, Robert Brink, Gulbu Uzel, Elissa K Deenick, Stuart G Tangye
Gain-of-function (GOF) mutations in PIK3CD , encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery...
July 17, 2018: Journal of Experimental Medicine
Diego Dibitetto, Mattia La Monica, Matteo Ferrari, Federica Marini, Achille Pellicioli
Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the kinetic of formation and 5'-end nucleolytic processing of Cas9-induced DSB in different human cells lines. Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken into consideration to improve Cas9-mediated technology...
August 2018: DNA Repair
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