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https://www.readbyqxmd.com/read/28534836/differentiation-and-structure-in-sulfolobus-islandicus-rod-shaped-virus-populations
#1
Maria A Bautista, Jesse A Black, Nicholas D Youngblut, Rachel J Whitaker
In the past decade, molecular surveys of viral diversity have revealed that viruses are the most diverse and abundant biological entities on Earth. In culture, however, most viral isolates that infect microbes are represented by a few variants isolated on type strains, limiting our ability to study how natural variation affects virus-host interactions in the laboratory. We screened a set of 137 hot spring samples for viruses that infect a geographically diverse panel of the hyperthemophilic crenarchaeon Sulfolobus islandicus...
May 19, 2017: Viruses
https://www.readbyqxmd.com/read/28534478/complementary-information-derived-from-crispr-cas9-mediated-gene-deletion-and-suppression
#2
Joseph Rosenbluh, Han Xu, William Harrington, Stanley Gill, Xiaoxing Wang, Francisca Vazquez, David E Root, Aviad Tsherniak, William C Hahn
CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes...
May 23, 2017: Nature Communications
https://www.readbyqxmd.com/read/28533524/enhancing-the-genome-editing-toolbox-genome-wide-crispr-arrayed-libraries
#3
Emmanouil Metzakopian, Alex Strong, Vivek Iyer, Alex Hodgkins, Konstantinos Tzelepis, Liliana Antunes, Mathias J Friedrich, Qiaohua Kang, Teresa Davidson, Jacob Lamberth, Christina Hoffmann, Gregory D Davis, George S Vassiliou, William C Skarnes, Allan Bradley
CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers...
May 22, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28533066/an-efficient-tool-for-metabolic-pathway-construction-and-gene-integration-for-aspergillus-niger
#4
Parveen Sarkari, Hans Marx, Marzena L Blumhoff, Diethard Mattanovich, Michael Sauer, Matthias G Steiger
Metabolic engineering requires functional genetic tools for easy and quick generation of multiple pathway variants. A genetic engineering toolbox for A. niger is presented, which facilitates the generation of strains carrying heterologous expression cassettes at a defined genetic locus. The system is compatible with Golden Gate cloning, which facilitates the DNA construction process and provides high design flexibility. The integration process is mediated by a CRISPR/Cas9 strategy involving the cutting of both the genetic integration locus (pyrG) as well as the integrating plasmid...
May 4, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28523286/synthetic-lethality-screens-using-rnai-in-combination-with-crispr-based-knockout-in-drosophila-cells
#5
Benjamin E Housden, Hilary E Nicholson, Norbert Perrimon
A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi...
February 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28515773/building-a-multipurpose-insertional-mutant-library-for-forward-and-reverse-genetics-in-chlamydomonas
#6
Xi Cheng, Gai Liu, Wenting Ke, Lijuan Zhao, Bo Lv, Xiaocui Ma, Nannan Xu, Xiaoling Xia, Xuan Deng, Chunlei Zheng, Kaiyao Huang
BACKGROUND: The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive...
2017: Plant Methods
https://www.readbyqxmd.com/read/28499832/versatile-and-precise-gene-targeting-strategies-for-functional-studies-in-mammalian-cell-lines
#7
REVIEW
M Wassef, A Luscan, A Battistella, S Le Corre, H Li, M R Wallace, M Vidaud, R Margueron
The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e...
May 10, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28494239/crispr-cas9-screens-reveal-epstein-barr-virus-transformed-b-cell-host-dependency-factors
#8
Yijie Ma, Michael J Walsh, Katharina Bernhardt, Camille W Ashbaugh, Stephen J Trudeau, Isabelle Y Ashbaugh, Sizun Jiang, Chang Jiang, Bo Zhao, David E Root, John G Doench, Benjamin E Gewurz
Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify host dependency factors resulting from these EBV+, B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival...
May 10, 2017: Cell Host & Microbe
https://www.readbyqxmd.com/read/28490437/high-throughput-crispri-phenotyping-identifies-new-essential-genes-in-streptococcus-pneumoniae
#9
Xue Liu, Clement Gallay, Morten Kjos, Arnau Domenech, Jelle Slager, Sebastiaan P van Kessel, Kèvin Knoops, Robin A Sorg, Jing-Ren Zhang, Jan-Willem Veening
Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%)...
May 10, 2017: Molecular Systems Biology
https://www.readbyqxmd.com/read/28487920/elucidating-drug-targets-and-mechanisms-of-action-by-genetic-screens-in-mammalian-cells
#10
Martin Kampmann
Phenotypic screening is a powerful approach to discover small molecules with desired effects on biological systems, which can then be developed into therapeutic drugs. The identification of the target and mechanism of action of compounds discovered in phenotypic screens remains a major challenge. This feature article describes the use of genetic tools to reveal drug targets and mechanisms in mammalian cells. Until recently, RNA interference was the method of choice for such studies. Here, we highlight very recent additions to the genetic toolkit in mammalian cells, including CRISPR, CRISPR interference, and CRISPR activation, and illustrate their usefulness for drug target identification...
May 10, 2017: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/28487703/heritable-genomic-fragment-deletions-and-small-indels-in-the-putative-engase-gene-induced-by-crispr-cas9-in-barley
#11
Eszter Kapusi, Maria Corcuera-Gómez, Stanislav Melnik, Eva Stoger
Targeted genome editing with the CRISPR/Cas9 system has been used extensively for the selective mutation of plant genes. Here we used CRISPR/Cas9 to disrupt the putative barley (Hordeum vulgare cv. "Golden Promise") endo-N-acetyl-β-D-glucosaminidase (ENGase) gene. Five single guide RNAs (sgRNAs) were designed for different target sites in the upstream part of the ENGase coding region. Targeted fragment deletions were induced by co-bombarding selected combinations of sgRNA with wild-type cas9 using separate plasmids, or by co-infection with separate Agrobacterium tumefaciens cultures...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28481362/genetic-interaction-mapping-in-mammalian-cells-using-crispr-interference
#12
Dan Du, Assen Roguev, David E Gordon, Meng Chen, Si-Han Chen, Michael Shales, John Paul Shen, Trey Ideker, Prashant Mali, Lei S Qi, Nevan J Krogan
We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation...
May 8, 2017: Nature Methods
https://www.readbyqxmd.com/read/28480303/a-one-step-pcr-based-assay-to-evaluate-the-efficiency-and-precision-of-genomic-dna-editing-tools
#13
Diego Germini, Yara Bou Saada, Tatiana Tsfasman, Kristina Osina, Chloé Robin, Nikolay Lomov, Mikhail Rubtsov, Nikolajs Sjakste, Mar Lipinski, Yegor Vassetzky
Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease...
June 16, 2017: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/28478951/a-crispr-approach-to-neurodegenerative-diseases
#14
Martin Kampmann
A major barrier to developing effective therapies for neurodegenerative diseases is our incomplete understanding of the underlying cellular mechanisms. Genetic screens in human-induced pluripotent stem cell-derived neurons can elucidate such mechanisms. Genome-wide screens using CRISPR interference and CRISPR activation provide complementary biological insights and may reveal potential therapeutic targets.
May 4, 2017: Trends in Molecular Medicine
https://www.readbyqxmd.com/read/28478735/gene-therapy-of-adult-neuronal-ceroid-lipofuscinoses-with-crispr-cas9-in-zebrafish
#15
Xiaomin Yao, Xiaowei Liu, Yaguang Zhang, Yuhao Li, Chengjian Zhao, Shaohua Yao, Yu-Quan Wei
Adult-Onset Neuronal Ceroid Lipofuscinosis (ANCL), one of the neuronal ceroid lipofuscinosis (NCLs), is an inherited neurodegenerative disorder with progressive neuronal dysfunction. Recently, mutations in DNAJC5 gene that encodes Cysteine-String protein Alpha (CSPα) have been reported to be associated with familial Autosomal-Dominant ANCL (AD-ANCL). Here, we constructed an ANCL transgenic zebrafish model expressing human mutant DNAJC5 (mDNAJC5) gene under the control of a zebrafish neuron-specific promoter...
May 6, 2017: Human Gene Therapy
https://www.readbyqxmd.com/read/28474669/genome-scale-measurement-of-off-target-activity-using-cas9-toxicity-in-high-throughput-screens
#16
David W Morgens, Michael Wainberg, Evan A Boyle, Oana Ursu, Carlos L Araya, C Kimberly Tsui, Michael S Haney, Gaelen T Hess, Kyuho Han, Edwin E Jeng, Amy Li, Michael P Snyder, William J Greenleaf, Anshul Kundaje, Michael C Bassik
CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin...
May 5, 2017: Nature Communications
https://www.readbyqxmd.com/read/28474250/single-cell-crispr-screening-in-drug-resistance
#17
EDITORIAL
William Wang, Xiangdong Wang
No abstract text is available yet for this article.
June 2017: Cell Biology and Toxicology
https://www.readbyqxmd.com/read/28460414/crispr-editing-technology-in-biological-and-biomedical-investigation
#18
Martyn K White, Rafal Kaminski, Won-Bin Young, Pamela C Roehm, Kamel Khalili
The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to confer provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation...
April 29, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28459458/circle-seq-a-highly-sensitive-in-vitro-screen-for-genome-wide-crispr-cas9-nuclease-off-targets
#19
Shengdar Q Tsai, Nhu T Nguyen, Jose Malagon-Lopez, Ved V Topkar, Martin J Aryee, J Keith Joung
Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations...
May 1, 2017: Nature Methods
https://www.readbyqxmd.com/read/28459451/generation-of-inner-ear-organoids-containing-functional-hair-cells-from-human-pluripotent-stem-cells
#20
Karl R Koehler, Jing Nie, Emma Longworth-Mills, Xiao-Ping Liu, Jiyoon Lee, Jeffrey R Holt, Eri Hashino
The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate...
May 1, 2017: Nature Biotechnology
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