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https://www.readbyqxmd.com/read/28646085/generation-of-a-collection-of-mutant-tomato-lines-using-pooled-crispr-libraries
#1
Thomas B Jacobs, Ning Zhang, Dhruv Patel, Gregory B Martin
The high efficiency of CRIPSR-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato, collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated gRNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted...
June 23, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28643249/genome-editing-of-mouse-by-cytoplasmic-injection
#2
Takuro Horii, Izuho Hatada
CRISPR/Cas enables rapid production of genome-edited animals. The Cas9/gRNA component can be introduced to fertilized eggs in several ways. Here, we provide an instructional guide for the generation of knockout mice by cytoplasmic injection using in vitro transcribed Cas9 and gRNA.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28643248/computational-prediction-of-crispr-cas9-target-sites-reveals-potential-off-target-risks-in-human-and-mouse
#3
Qingbo Wang, Kumiko Ui-Tei
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a prominent genome engineering technology. In the CRISPR/Cas system, the RNA-guided endonuclease Cas protein introduces a DNA double-stranded break at the genome position recognized by a guide RNA (gRNA) based on complementary base-pairing of about 20-nucleotides in length. The 8- or 12-mer gRNA sequence in the proximal region is especially important for target recognition, and the genes with sequence complementarity to such regions are often disrupted...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28640612/crispr-cas9-based-genome-editing-for-disease-modeling-and-therapy-challenges-and-opportunities-for-nonviral-delivery
#4
Hong-Xia Wang, Mingqiang Li, Ciaran M Lee, Syandan Chakraborty, Hae-Won Kim, Gang Bao, Kam W Leong
Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors...
June 22, 2017: Chemical Reviews
https://www.readbyqxmd.com/read/28630892/disruption-of-the-axonal-trafficking-of-tyrosine-hydroxylase-mrna-impairs-catecholamine-biosynthesis-in-the-axons-of-sympathetic-neurons
#5
Armaz Aschrafi, Anthony E Gioio, Lijin Dong, Barry B Kaplan
Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3'untranslated region (3'UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal...
May 2017: ENeuro
https://www.readbyqxmd.com/read/28624219/crispr-cas9-mediated-three-nucleotide-insertion-corrects-a-deletion-mutation-in-mrp1-abcc1-and-restores-its-proper-folding-and-function
#6
Qinqin Xu, Yue-Xian Hou, Xiu-Bao Chang
A three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletion mutant in another ABCC family member, multidrug resistance-associated protein (MRP1)/ABCC1, to determine whether CRISPR-Cas9-mediated recombination can safely and efficiently knock in three-nucleotide to correct the mutation...
June 16, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28624187/correction-of-the-exon-2-duplication-in-dmd-myoblasts-by-a-single-crispr-cas9-system
#7
Annalisa Lattanzi, Stephanie Duguez, Arianna Moiani, Araksya Izmiryan, Elena Barbon, Samia Martin, Kamel Mamchaoui, Vincent Mouly, Francesco Bernardi, Fulvio Mavilio, Matteo Bovolenta
Exonic duplications account for 10%-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection...
June 16, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28610973/a-two-plasmid-inducible-crispr-cas9-genome-editing-tool-for-clostridium-acetobutylicum
#8
François Wasels, Jennifer Jean-Marie, Florent Collas, Ana M López-Contreras, Nicolas Lopes Ferreira
CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid...
June 10, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28603751/production-of-guide-rnas-in-vitro-and-in-vivo-for-crispr-using-ribozymes-and-rna-polymerase-ii-promoters
#9
Tao Zhang, Yangbin Gao, Rongchen Wang, Yunde Zhao
CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.
February 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28581909/development-of-crispr-cas9-mediated-virus-resistance-in-agriculturally-important-crops
#10
Surender Khatodia, Kirti Bhatotia, Narendra Tuteja
Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) system of targeted genome editing has already revolutionized the plant science research. This is a RNA guided programmable endonuclease based system composed of 2 components, the Cas9 nuclease and an engineered guide RNA targeting any DNA sequence of the form N20-NGG for novel genome editing applications. The CRISPR/Cas9 technology of targeted genome editing has been recently applied for imparting virus resistance in plants...
May 4, 2017: Bioengineered
https://www.readbyqxmd.com/read/28558198/engineered-crispr-systems-for-next-generation-gene-therapies
#11
Michael Pineda, Farzaneh Moghadam, Mo R Ebrahimkhani, Samira Kiani
An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence...
June 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28557624/rna-guided-activation-of-pluripotency-genes-in-human-fibroblasts
#12
Kai Xiong, Yan Zhou, Kristian Aabo Blichfeld, Poul Hyttel, Lars Bolund, Kristine Karla Freude, Yonglun Luo
Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts)...
June 2017: Cellular Reprogramming
https://www.readbyqxmd.com/read/28542388/targeted-dna-methylation-in-pericentromeres-with-genome-editing-based-artificial-dna-methyltransferase
#13
Taiga Yamazaki, Yu Hatano, Tetsuya Handa, Sakiko Kato, Kensuke Hoida, Rui Yamamura, Takashi Fukuyama, Takayuki Uematsu, Noritada Kobayashi, Hiroshi Kimura, Kazuo Yamagata
To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI...
2017: PloS One
https://www.readbyqxmd.com/read/28541304/digital-logic-circuits-in-yeast-with-crispr-dcas9-nor-gates
#14
Miles W Gander, Justin D Vrana, William E Voje, James M Carothers, Eric Klavins
Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses...
May 25, 2017: Nature Communications
https://www.readbyqxmd.com/read/28535252/trypanosome-rna-editing-mediator-complex-proteins-have-distinct-functions-in-grna-utilization
#15
Rachel M Simpson, Andrew E Bruno, Runpu Chen, Kaylen Lott, Brianna L Tylec, Jonathan E Bard, Yijun Sun, Michael J Buck, Laurie K Read
Uridine insertion/deletion RNA editing is an essential process in kinetoplastid parasites whereby mitochondrial mRNAs are modified through the specific insertion and deletion of uridines to generate functional open reading frames, many of which encode components of the mitochondrial respiratory chain. The roles of numerous non-enzymatic editing factors have remained opaque given the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus roles of individual proteins...
May 23, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28531199/mir-145-mediates-zebrafish-hepatic-outgrowth-through-progranulin-a-signaling
#16
Ya-Wen Li, Keng-Yu Chiang, Yen-Hsing Li, Sung-Yu Wu, Wangta Liu, Chia-Ray Lin, Jen-Leih Wu
MicroRNAs (miRs) are mRNA-regulatory molecules that fine-tune gene expression and modulate both processes of development and tumorigenesis. Our previous studies identified progranulin A (GrnA) as a growth factor which induces zebrafish hepatic outgrowth through MET signaling. We also found that miR-145 is one of potential fine-tuning regulators of GrnA involved in embryonic hepatic outgrowth. The low level of miR-145 seen in hepatocarinogenesis has been shown to promote pathological liver growth. However, little is known about the regulatory mechanism of miR-145 in embryonic liver development...
2017: PloS One
https://www.readbyqxmd.com/read/28495970/high-throughput-biochemical-profiling-reveals-sequence-determinants-of-dcas9-off-target-binding-and-unbinding
#17
Evan A Boyle, Johan O L Andreasson, Lauren M Chircus, Samuel H Sternberg, Michelle J Wu, Chantal K Guegler, Jennifer A Doudna, William J Greenleaf
The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM)...
May 23, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28492550/txnip-regulates-mitophagy-in-retinal-m%C3%A3-ller-cells-under-high-glucose-conditions-implications-for-diabetic-retinopathy
#18
Takhellambam Swornalata Devi, Mallika Somayajulu, Renu Anjan Kowluru, Lalit Pukhrambam Singh
Thioredoxin-interacting protein (TXNIP) is involved in oxidative stress and apoptosis in diabetic retinopathy. However, the role of TXNIP in the removal of damaged mitochondria (MT) via mitophagy, a process of macroautophagy, remains unexplored. Here we investigate the associated cellular and molecular mechanisms underlying mitophagy in retinal cells under diabetic conditions. For this, we maintained a rat Müller cell line (rMC1) under high-glucose (25 mM, HG) or low-glucose (5.5 mM, LG) condition for 5 days...
May 11, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28462777/synthetically-modified-guide-rna-and-donor-dna-are-a-versatile-platform-for-crispr-cas9-engineering
#19
Kunwoo Lee, Vanessa A Mackley, Anirudh Rao, Anthony T Chong, Mark A Dewitt, Jacob E Corn, Niren Murthy
Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5' fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA...
May 2, 2017: ELife
https://www.readbyqxmd.com/read/28456574/suppression-of-epstein-barr-virus-dna-load-in-latently-infected-nasopharyngeal-carcinoma-cells-by-crispr-cas9
#20
Kit-San Yuen, Zhong-Min Wang, Nok-Hei Mickey Wong, Zhi-Qian Zhang, Tsz-Fung Cheng, Wai-Yin Lui, Chi-Ping Chan, Dong-Yan Jin
Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%...
April 27, 2017: Virus Research
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