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CRISPR selection

Aikaterini N Tsouka, Constantinos C Tellis, Alexandros D Tselepis
Protein Convertase Subtilisin/Kexin type 9 (PCSK9) is a serine protease primarily expressed in the liver, which represents the main source of the plasma enzyme. The best characterized function of PCSK9 relates to the binding to low-density lipoprotein receptor (LDL-R) in hepatocytes, increasing its endosomal and lysosomal degradation. This results in the inhibition of LDL-R recycling to the cell surface and therefore the reduction of the hepatic uptake of LDL, leading to the increase in plasma levels of LDL-cholesterol, a major risk factor of cardiovascular diseases (CVD)...
October 10, 2018: Current Pharmaceutical Design
Lu Long, Dan-Dan Guo, Wei Gao, Wen-Wen Yang, Li-Pan Hou, Xiao-Nan Ma, Yu-Chen Miao, Jose Ramon Botella, Chun-Peng Song
Background: When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome...
2018: Plant Methods
Tiffany Leidy-Davis, Kai Cheng, Leslie O Goodwin, Judith L Morgan, Wen Chun Juan, Xavier Roca, S Tiong Ong, David E Bergstrom
Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2...
October 9, 2018: Scientific Reports
Ashley Rg Libby, David A Joy, Po-Lin So, Mohammad A Mandegar, Jonathon M Muncie, Federico N Mendoza-Camacho, Valerie M Weaver, Bruce R Conklin, Todd C McDevitt
Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference...
October 9, 2018: ELife
Avery C Rossidis, John D Stratigis, Alexandra C Chadwick, Heather A Hartman, Nicholas J Ahn, Haiying Li, Kshitiz Singh, Barbara E Coons, Li Li, Wenjian Lv, Philip W Zoltick, Deepthi Alapati, William Zacharias, Rajan Jain, Edward E Morrisey, Kiran Musunuru, William H Peranteau
In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting...
October 2018: Nature Medicine
Marcela Vilarino, Fabian Patrik Suchy, Sheikh Tamir Rashid, Helen Lindsay, Juan Reyes, Bret Roberts McNabb, Talitha van der Meulen, Mark O Huising, Hiromitsu Nakauchi, Pablo Juan Ross
The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep...
October 3, 2018: Transgenic Research
Sonali Majumdar, Michael P Terns
Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA...
October 3, 2018: Extremophiles: Life Under Extreme Conditions
Thomas W Kirchner, Markus Niehaus, Kim L Rössig, Timo Lauterbach, Marco Herde, Helge Küster, Manfred K Schenk
Formation of longer root hairs under limiting phosphate (P) conditions can increase the inorganic P (Pi) uptake. Here, regulatory candidate genes for Pi deficiency-induced root hair growth were identified by comparison of massive analysis of cDNA ends (MACE) provided expression profiles of two Brassica carinata cultivars (cv.) differing in their root hair response to Pi deficiency: cv. Bale develops longer root hairs under Pi deficiency, but not cv. Bacho. A split-root experiment was conducted for the differentiation between locally and systemically regulated genes...
2018: Frontiers in Plant Science
Mateus S Terceti, Ana Vences, Xosé M Matanza, Inger Dalsgaard, Karl Pedersen, Carlos R Osorio
The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively...
2018: Frontiers in Microbiology
Stefanie L Morgan, Erin Y Chang, Natasha C Mariano, Abel Bermudez, Nicole L Arruda, Fanting Wu, Yunhai Luo, Gautam Shankar, Star K Huynh, Chiao-Chain Huang, Sharon J Pitteri, Kevin C Wang
Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions...
September 14, 2018: Journal of Visualized Experiments: JoVE
Annamaria Mocciaro, Theodore L Roth, Hayley M Bennett, Magali Soumillon, Abhik Shah, Joseph Hiatt, Kevin Chapman, Alexander Marson, Gregory Lavieu
Despite improvements in the CRISPR molecular toolbox, identifying and purifying properly edited clones remains slow, laborious, and low-yield. Here, we establish a method to enable clonal isolation, selection, and expansion of properly edited cells, using OptoElectroPositioning technology for single-cell manipulation on a nanofluidic device. Briefly, after electroporation of primary T cells with CXCR4 -targeting Cas9 ribonucleoproteins, single T cells are isolated on a chip and expanded into colonies. Phenotypic consequences of editing are rapidly assessed on-chip with cell-surface staining for CXCR4...
2018: Communications biology
Mahsa Zarei, Shruti Lal, Ali Vaziri-Gohar, Kevin O'Hayer, Venugopal Gunda, Pankaj K Singh, Jonathan R Brody, Jordan M Winter
Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic enzyme in human malignancy. A heterozygous genetic alteration, arginine 132, promotes the conversion of α-ketoglutarate to D-2-hydroxyglutarate (2-HG). While pharmacologic inhibitors of mutant IDH1 are promising, resistance mechanisms to targeted therapy are not understood. Additionally, the role of wild-type IDH1 (WT.IDH1) in cancer requires further study. Recently it was observed that the regulatory RNA-binding protein, HuR (ELAVL1), protects nutrient deprived cancer cells without IDH1 mutations, by stabilizing WT...
September 28, 2018: Molecular Cancer Research: MCR
Bridget L Menasche, Lauren Crisman, Daniel R Gulbranson, Eric M Davis, Haijia Yu, Jingshi Shen
About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing...
September 28, 2018: Current Protocols in Cell Biology
Uwe Fiebig, Konrad Fischer, Andrea Bähr, Carolin Runge, Angelika Schnieke, Eckhard Wolf, Joachim Denner
BACKGROUND: Porcine endogenous retroviruses (PERVs) may pose a risk of xenotransplantation using porcine cells, tissues, or organs. PERVs are integrated in the genome of all pigs, and some can infect certain human cells. The copy number of PERVs in different pig breeds has been determined by using different methods, with varying results. METHODS: To determine the PERV copy number in pig cell lines and in animals, a new method, droplet digital polymerase chain reaction (ddPCR) was used...
July 2018: Xenotransplantation
Xiaohong Zhang, Yanqing Qian, Fan Li, Songhua Bei, Meiyi Li, Li Feng
LIM homeobox transcription factor 1, alpha (LMX1A) is downregulated in human gastric cancer (GC), functioning as a tumor suppressor. The current study aims to identify specific microRNA that can regulate LMX1A expression. By sequence analysis of LMX1A mRNA 3'-untranslated region (3'-UTR), we show that microRNA-9 (miR-9) putatively targets human LMX1A. In established (AGS cells) and primary human GC cells, ectopic overexpression of miR-9 by a lentiviral construct decreased LMX1A 3'-UTR activity, causing LMX1A mRNA and protein downregulation...
September 24, 2018: Biochemical and Biophysical Research Communications
Abraham Peele Karlapudi, Venkateswarulu T C, Jahnavi Tammineedi, Krupanidhi Srirama, Lohit Kanumuri, Vidya Prabhakar Kodali
CRISPR genome editing utilizes Cas9 nuclease and single guide RNA (sgRNA), which directs the nuclease to a specific site in the genome and makes a double-stranded break (DSB). Design of sgRNA for CRISPR-Cas targeting, and to promote CRISPR adaptation, uses a regulatory mechanism that ensures maximum CRISPR-Cas9 system functions when a bacterial population is at highest risk of phage infection. Acinetobacter baumannii is the most regularly identified gram-negative bacterium infecting patients. Recent reports have demonstrated that the extent of diseases caused by A...
June 2018: Genes & Diseases
Haruyasu Hamada, Yuelin Liu, Yozo Nagira, Ryuji Miki, Naoaki Taoka, Ryozo Imai
The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds...
September 26, 2018: Scientific Reports
A K Sharma, M Nymark, T Sparstad, A M Bones, P Winge
The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells...
September 26, 2018: Scientific Reports
Rachel M Guerra, Gregory H Bird, Edward P Harvey, Neekesh V Dharia, Kyle J Korshavn, Michelle S Prew, Kimberly Stegmaier, Loren D Walensky
Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells...
September 25, 2018: Cell Reports
Hai Yang, Rui Chen, Quan Wang, Qiang Wei, Ying Ji, Guangze Zheng, Xue Zhong, Nancy J Cox, Bingshan Li
Motivation: Given the complexity of genome regions, prioritize the functional effects of noncoding variants remains a challenge. Although several frameworks have been proposed for the evaluation of the functionality of noncoding variants, most of them used "black boxes" methods that simplify the task as the pathogenicity/benign classification problem, which ignores the distinct regulatory mechanisms of variants and leads to less desirable performance. In this study, we developed DVAR, an unsupervised framework that leverage various biochemical and evolutionary evidence to distinguish the gene regulatory categories of variants and assess their comprehensive functional impact simultaneously...
September 26, 2018: Bioinformatics
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