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https://www.readbyqxmd.com/read/28432350/enhancing-the-throughput-and-multiplexing-capabilities-of-next-generation-sequencing-for-efficient-implementation-of-pooled-shrna-and-crispr-screens
#1
Md Fahmid Islam, Atsushi Watanabe, Lai Wong, Conor Lazarou, Frederick S Vizeacoumar, Omar Abuhussein, Wayne Hill, Maruti Uppalapati, C Ronald Geyer, Franco J Vizeacoumar
Next generation sequencing is becoming the method of choice for functional genomic studies that use pooled shRNA or CRISPR libraries. A key challenge in sequencing these mixed-oligo libraries is that they are highly susceptible to hairpin and/or heteroduplex formation. This results in polyclonal, low quality, and incomplete reads and reduces sequencing throughput. Unfortunately, this challenge is significantly magnified in low-to-medium throughput bench-top sequencers as failed reads significantly perturb the maximization of sequence coverage and multiplexing capabilities...
April 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28432338/crispr-cas9-mediated-inactivation-of-argonaute-2-reveals-its-differential-involvement-in-antiviral-responses
#2
Márta Ludman, József Burgyán, Károly Fátyol
RNA silencing constitutes an important antiviral mechanism in plants. Small RNA guided Argonaute proteins fulfill essential role in this process by acting as executors of viral restriction. Plants encode multiple Argonaute proteins of which several exhibit antiviral activities. A recent addition to this group is AGO2. Its involvement in antiviral responses is established predominantly by studies employing mutants of Arabidopsis thaliana. In the virological model plant, Nicotiana benthamiana, the contribution of AGO2 to antiviral immunity is much less certain due to the lack of appropriate genetic mutants...
April 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28432220/mir-155-promotes-flt3-itd-induced-myeloproliferative-disease-through-inhibition-of-the-interferon-response
#3
Jared A Wallace, Dominique A Kagele, Anna M Eiring, Carissa N Kim, Ruozhen Hu, Marah C Runtsch, Margaret Alexander, Thomas B Huffaker, Soh-Hyun Lee, Ami B Patel, Timothy L Mosbruger, Warren Voth, Dinesh S Rao, Rodney R Miles, June L Round, Michael W Deininger, Ryan M O'Connell
FLT3-ITD(+) AML accounts for approximately 25% of all AML cases, and is a subtype that carries a poor prognosis. miR-155 is specifically overexpressed in FLT3-ITD(+) AML compared to FLT3-WT AML, and is critical for the growth of FLT3-ITD(+) AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we utilized a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood...
April 21, 2017: Blood
https://www.readbyqxmd.com/read/28431253/crispr-based-technologies-for-the-manipulation-of-eukaryotic-genomes
#4
Alexis C Komor, Ahmed H Badran, David R Liu
No abstract text is available yet for this article.
April 20, 2017: Cell
https://www.readbyqxmd.com/read/28431233/a-compendium-of-rna-binding-proteins-that-regulate-microrna-biogenesis
#5
Thomas Treiber, Nora Treiber, Uwe Plessmann, Simone Harlander, Julia-Lisa Daiß, Norbert Eichner, Gerhard Lehmann, Kevin Schall, Henning Urlaub, Gunter Meister
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28431230/structural-basis-for-guide-rna-processing-and-seed-dependent-dna-targeting-by-crispr-cas12a
#6
Daan C Swarts, John van der Oost, Martin Jinek
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28430577/synthetic-lethal-interaction-between-the-tumour-suppressor-stag2-and-its-paralog-stag1
#7
Lorena Benedetti, Matteo Cereda, LeeAnn Monteverde, Nikita Desai, Francesca D Ciccarelli
Cohesin is a multi-protein complex that tethers sister chromatids during mitosis and mediates DNA repair, genome compartmentalisation and regulation of gene expression. Cohesin subunits frequently acquire cancer loss-of-function alterations and act as tumour suppressors in several tumour types. This has led to increased interest in cohesin as potential target in anti-cancer therapy. Here we show that the loss-of-function of STAG2, a core component of cohesin and an emerging tumour suppressor, leads to synthetic dependency of mutated cancer cells on its paralog STAG1...
April 5, 2017: Oncotarget
https://www.readbyqxmd.com/read/28430356/crispr-cas9-an-rna-guided-highly-precise-synthetic-tool-for-plant-genome-editing
#8
REVIEW
Yeliz Demirci, Baohong Zhang, Turgay Unver
CRISPR/Cas 9 is a newly developed and naturally occurred genome editing tool, which is originally used by bacteria for immune defence. In the past years, it has been quickly employed and modified to precisely edit genome sequences in both plants and animals. Compared with the well-developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas 9 has lots of advantages, including easier to design and implement, higher targeting efficiency, and less expensive. Thus, it is becoming one of the most powerful tools for knockout of an individual gene as well as insertion of one gene and/or control of gene transcription...
April 21, 2017: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/28429771/crispr-cas9-mediated-genome-editing-in-one-blastomere-of-two-cell-embryos-reveals-a-novel-tet3-function-in-regulating-neocortical-development
#9
Lingbo Wang, Min-Yin Li, Chao Qu, Wan-Ying Miao, Qi Yin, Jiaoyang Liao, Hua-Teng Cao, Min Huang, Kai Wang, Erwei Zuo, Guangdun Peng, Shu-Xin Zhang, Guodong Chen, Qing Li, Ke Tang, Qian Yu, Zhoujie Li, Catherine Cl Wong, Guoliang Xu, Naihe Jing, Xiang Yu, Jinsong Li
Studying the early function of essential genes is an important and challenging problem in developmental biology. Here, we established a method for rapidly inducing CRISPR-Cas9-mediated mutations in one blastomere of two-cell stage embryos, termed 2-cell embryo-CRISPR-Cas9 injection (2CC), to study the in vivo function of essential (or unknown) genes in founder chimeric mice. By injecting both Cre mRNA and CRISPR-Cas9 targeting the gene of interest into fluorescent reporter mice, the 2CC method can trace both wild-type and mutant cells at different developmental stages, offering internal control for phenotypic analyses of mutant cells...
April 21, 2017: Cell Research
https://www.readbyqxmd.com/read/28429769/gene-and-mutation-independent-therapy-via-crispr-cas9-mediated-cellular-reprogramming-in-rod-photoreceptors
#10
Jie Zhu, Chang Ming, Xin Fu, Yaou Duan, Duc Anh Hoang, Jeffrey Rutgard, Runze Zhang, Wenqiu Wang, Rui Hou, Daniel Zhang, Edward Zhang, Charlotte Zhang, Xiaoke Hao, Wenjun Xiong, Kang Zhang
No abstract text is available yet for this article.
April 21, 2017: Cell Research
https://www.readbyqxmd.com/read/28429283/use-of-hormones-tissue-factors-and-bioengineering-in-the-management-of-hypospadias
#11
Aparajita Mitra, Yogesh Kumar Sarin
Hypospadiology is a rapidly evolving field. Progress in the understanding of how hormonal therapy affects the growth of the phallus has allowed surgeons to optimize the tissues for surgery. But conflicting data from a number of studies and a lack of consensus on drugs, their dosing, mode of delivery and timing of use means that the creation of protocols is unlikely to happen in the near future. Nonetheless, there is a hope and the standardization of scientific reporting will make it easier to compare data at the global level...
April 21, 2017: Indian Journal of Pediatrics
https://www.readbyqxmd.com/read/28429203/regulating-the-new-a-consideration-of-crispr-and-approaches-to-professional-standards-of-practitioners-of-chinese-medicine-in-australia-and-accessing-the-ndis
#12
Barry R Furrow, Bernadette J Richards
No abstract text is available yet for this article.
April 20, 2017: Journal of Bioethical Inquiry
https://www.readbyqxmd.com/read/28428833/complete-genome-sequence-of-kosakonia-oryzae-type-strain-ola-51-t
#13
Yuanyuan Li, Shuying Li, Mingyue Chen, Guixiang Peng, Zhiyuan Tan, Qianli An
Strain Ola 51(T) (=LMG 24251(T) = CGMCC 1.7012(T)) is the type strain of the species Kosakonia oryzae and was isolated from surface-sterilized roots of the wild rice species Oryza latifolia grown in Guangdong, China. Here we summarize the features of the strain Ola 51(T) and describe its complete genome sequence. The genome contains one circular chromosome of 5,303,342 nucleotides with 54.01% GC content, 4773 protein-coding genes, 16 rRNA genes, 76 tRNA genes, 13 ncRNA genes, 48 pseudo genes, and 1 CRISPR array...
2017: Standards in Genomic Sciences
https://www.readbyqxmd.com/read/28428262/uncoupling-neurogenic-gene-networks-in-the-drosophila-embryo
#14
William A Rogers, Yogesh Goyal, Kei Yamaya, Stanislav Y Shvartsman, Michael S Levine
The EGF signaling pathway specifies neuronal identities in the Drosophila embryo by regulating developmental patterning genes such as intermediate neurons defective (ind). EGFR is activated in the ventral midline and neurogenic ectoderm by the Spitz ligand, which is processed by the Rhomboid protease. CRISPR/Cas9 was used to delete defined rhomboid enhancers mediating expression at each site of Spitz processing. Surprisingly, the neurogenic ectoderm, not the ventral midline, was found to be the dominant source of EGF patterning activity...
April 20, 2017: Genes & Development
https://www.readbyqxmd.com/read/28428254/a-novel-physiological-role-for-arf1-in-the-formation-of-bi-directional-tubules-from-the-golgi
#15
Francesca Bottanelli, Nicole Kilian, Andreas M Ernst, Felix Rivera-Molina, Lena K Schroeder, Emil B Kromann, Mark D Lessard, Roman S Erdmann, Alanna Schepartz, David Baddeley, Joerg Bewersdorf, Derek Toomre, James E Rothman
Capitalizing on CRISPR/Cas9 gene editing techniques and super-resolution nanoscopy, we have explored the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Surprisingly, beside its well-established role in generating COPI vesicles, we find that ARF1 is additionally involved in the formation of long (∼ 3 μm), thin (∼ 110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins Coatomer (COPI) and Clathrin...
April 20, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28427715/multiplexed-dynamic-imaging-of-genomic-loci-by-combined-crispr-imaging-and-dna-sequential-fish
#16
Yodai Takei, Sheel Shah, Sho Harvey, Lei S Qi, Long Cai
Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call "track first and identify later" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization...
April 17, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28427330/pan-genome-and-crispr-analyses-of-the-bacterial-fish-pathogen-moritella-viscosa
#17
Christian Karlsen, Erik Hjerde, Terje Klemetsen, Nils Peder Willassen
BACKGROUND: Winter-ulcer Moritella viscosa infections continue to be a significant burden in Atlantic salmon (Salmo salar L.) farming. M. viscosa comprises two main clusters that differ in genetic variation and phenotypes including virulence. Horizontal gene transfer through acquisition and loss of mobile genetic elements (MGEs) is a major driving force of bacterial diversification. To gain insight into genomic traits that could affect sublineage evolution within this bacterium we examined the genome sequences of twelve M...
April 20, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28426962/evidence-for-mechanosensitive-channel-activity-of-tentonin-3-tmem150c
#18
Gyu-Sang Hong, Byeongjun Lee, Uhtaek Oh
Mechanosensation is essential for various physiological processes, and it is mediated by mechanotransduction channels. Recently, we reported that TMEM150C/Tentonin 3 (TTN3) confers mechanically activated currents with slow inactivation kinetics in several cell types, including dorsal root ganglion neurons (Hong et al., 2016). The accompanying Matters Arising by Dubin, Murthy, and colleagues confirms that naive heterologous cells demonstrate a mechanically activated current, but finds that this response is absent in CRISPR-Cas9 Piezo1 knockout cell lines and suggests that TTN3 is a modulator of Piezo1...
April 19, 2017: Neuron
https://www.readbyqxmd.com/read/28426961/endogenous-piezo1-can-confound-mechanically-activated-channel-identification-and-characterization
#19
Adrienne E Dubin, Swetha Murthy, Amanda H Lewis, Lucie Brosse, Stuart M Cahalan, Jörg Grandl, Bertrand Coste, Ardem Patapoutian
A gold standard for characterizing mechanically activated (MA) currents is via heterologous expression of candidate channels in naive cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of an MA channel partly based on a heterologous expression approach (Hong et al., 2016). In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao et al...
April 19, 2017: Neuron
https://www.readbyqxmd.com/read/28426791/correction-cctop-an-intuitive-flexible-and-reliable-crispr-cas9-target-prediction-tool
#20
Manuel Stemmer, Thomas Thumberger, Maria Del Sol Keyer, Joachim Wittbrodt, Juan L Mateo
[This corrects the article DOI: 10.1371/journal.pone.0124633.].
2017: PloS One
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