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https://www.readbyqxmd.com/read/28918056/the-therapeutic-potential-of-crispr-cas9-systems-in-oncogene-addicted-cancer-types-virally-driven-cancers-as-a-model-system
#1
REVIEW
Luqman Jubair, Nigel A J McMillan
The field of gene editing is undergoing unprecedented growth. The first ex vivo human clinical trial in China started in 2016, more than 1000 US patents have been filed, and there is exponential growth in publications. The ability to edit genes with high fidelity is promising for the development of new treatments for a range of diseases, particularly inherited conditions, infectious diseases, and cancers. For cancer, a major issue is the identification of driver mutations and oncogenes to target for therapeutic effect, and this requires the development of robust models with which to prove their efficacy...
September 15, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28917058/lineage-tracing-of-genome-edited-alleles-reveals-high-fidelity-axolotl-limb-regeneration
#2
Grant Parker Flowers, Lucas D Sanor, Craig M Crews
Salamanders are unparalleled among tetrapods in their ability to regenerate many structures, including entire limbs, and the study of this ability may provide insights into human regenerative therapies. The complex structure of the limb poses challenges to the investigation of the cellular and molecular basis of its regeneration. Using CRISPR/Cas, we genetically labelled unique cell lineages within the developing axolotl embryo and tracked the frequency of each lineage within amputated and fully regenerated limbs...
September 16, 2017: ELife
https://www.readbyqxmd.com/read/28915012/managing-the-sos-response-for-enhanced-crispr-cas-based-recombineering-in-e-coli-through-transient-inhibition-of-host-reca-activity
#3
Eirik Adim Moreb, Benjamin Hoover, Adam Yaseen, Nisakorn Valyasevi, Zoe Roecker, Romel Menacho-Melgar, Michael Lynch
Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well mutation rates through the induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants...
September 15, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28913801/targeted-mutagenesis-in-hexaploid-bread-wheat-using-the-talen-and-crispr-cas-systems
#4
Yanpeng Wang, Yuan Zong, Caixia Gao
The use of sequence-specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas9) have provided powerful reverse genetic approaches to the targeted modification of genomes in numerous organisms. Both systems have been employed to generate loss-of-function alleles in bread wheat, by targeting multiple and single copies of genes. Here we present protocols for modifying the wheat genome using the two systems. The protocols include the design of TALEN and CRISPR/Cas9 target sites and their construction, evaluation of their activities in protoplasts, transformation of plants, and mutation screening...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28911114/coupling-transcriptional-activation-of-crispr-cas-system-and-dna-repair-genes-by-csa3a-in-sulfolobus-islandicus
#5
Tao Liu, Zhenzhen Liu, Qing Ye, Saifu Pan, Xiaodi Wang, Yingjun Li, Wenfang Peng, Yunxiang Liang, Qunxin She, Nan Peng
CRISPR-Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system...
September 6, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28910979/crispr-cas9-based-efficient-genome-editing-in-staphylococcus-aureus
#6
Qi Liu, Yu Jiang, Lei Shao, Ping Yang, Bingbing Sun, Sheng Yang, Daijie Chen
Staphylococcus aureus is an important pathogenic bacterium prevalent in nosocomial infections and associated with high morbidity and mortality rates, which arise from the significant pathogenicity and multi-drug resistance. However, the typical genetic manipulation tools used to explore the relevant molecular mechanisms of S. aureus have multiple limitations: leaving a scar in the genome, comparatively low gene-editing efficiency, and prolonged experimental period. Here, we present a single-plasmid based on the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system which allows rapid and efficient chromosomal manipulation in S...
September 1, 2017: Acta Biochimica et Biophysica Sinica
https://www.readbyqxmd.com/read/28910631/specialized-weaponry-how-a-type-iii-a-crispr-cas-system-excels-at-combating-phages
#7
Ole Niewoehner, Martin Jinek
CRISPR-Cas-mediated defense against phage invaders usually requires recognition of short sequences, termed protospacer-adjacent motifs (PAMs), in phage DNA. In this issue of Cell Host & Microbe, Pyenson et al. (2017) show that the lack of a PAM requirement in some CRISPR-Cas systems prevents interference evasion and facilitates phage extinction.
September 13, 2017: Cell Host & Microbe
https://www.readbyqxmd.com/read/28905415/hyperactive-mtor-induces-neuroendocrine-differentiation-in-prostate-cancer-cell-with-concurrent-up-regulation-of-irf1
#8
Mayuko Kanayama, Toshiya Hayano, Michinori Koebis, Tatsuya Maeda, Yoko Tabe, Shigeo Horie, Atsu Aiba
BACKGROUND: Neuroendocrine-differentiated prostate cancer (NEPCa) is refractory to androgen deprivation therapy and shows a poor prognosis. The underlying mechanisms responsible for neuroendocrine differentiation (NED) are yet to be clarified. In this study, we investigated the role of mammalian target of rapamycin (mTOR) in NEPCa. METHODS: We utilized a gain-of-function analysis by establishing a human PCa LNCaP stable line that expresses hyperactive mTOR (LNCaP-mTOR)...
September 14, 2017: Prostate
https://www.readbyqxmd.com/read/28900693/complete-genome-sequence-of-geobacillus-thermodenitrificans-t12-a-potential-host-for-biotechnological-applications
#9
Martinus J A Daas, Bastienne Vriesendorp, Antonius H P van de Weijer, John van der Oost, Richard van Kranenburg
In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3...
September 12, 2017: Current Microbiology
https://www.readbyqxmd.com/read/28900076/-crispr-cas9-the-king-of-genome-editing-tools
#10
A V Bannikov, A V Lavrov
The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing...
July 2017: Molekuliarnaia Biologiia
https://www.readbyqxmd.com/read/28893837/crrnas-in-porphyromonas-gingivalis-crispr-cas-i-c-system
#11
Michal Burmistrz, Jose Ignacio Rodriguez Martinez, Daniel Krochmal, Dominika Staniec, Krzysztof Pyrc
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) system is unique to prokaryotes and provides the majority of bacteria and archaea with immunity against nucleic acids of foreign origin. crRNAs are the key element of this system, since they are responsible for its selectivity and effectiveness. Typical crRNAs consist of a spacer sequence flanked with 5' and 3' handles originating from repeat sequences that are important for recognition of these small RNAs by the Cas machinery...
September 11, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28892041/guide-bound-structures-of-an-rna-targeting-a-cleaving-crispr-cas13a-enzyme
#12
Gavin J Knott, Alexandra East-Seletsky, Joshua C Cofsky, James M Holton, Emeric Charles, Mitchell R O'Connell, Jennifer A Doudna
CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation...
September 11, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28888103/using-crispr-cas-systems-as-antimicrobials
#13
REVIEW
David Bikard, Rodolphe Barrangou
Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death...
September 5, 2017: Current Opinion in Microbiology
https://www.readbyqxmd.com/read/28886218/crispr-cpf1-enables-fast-and-simple-genome-editing-of-saccharomyces-cerevisiae
#14
René Verwaal, Nathalie Buiting-Wiessenhaan, Sacha Dalhuijsen, Johannes A Roubos
Cpf1 represents a novel single RNA-guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared to Cas9. We demonstrate the functionality of three Cpf1 orthologues - Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), Francisella novicida U112 (FnCpf1) - for genome editing of Saccharomyces cerevisiae. These Cpf1-based systems enable fast and reliable introduction of donor DNA on the genome using a two plasmid-based editing approach together with linear donor DNA...
September 8, 2017: Yeast
https://www.readbyqxmd.com/read/28879852/type-iii-crispr-cas-system-introduction-and-its-application-for-genetic-manipulations
#15
Tao Liu, Saifu Pan, Yingjun Li, Nan Peng, Qunxin She
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes provide adaptive immunity against invasion of foreign nucleic acids in archaea and bacteria. The system functions in three distinct stages: adaptation, biogenesis, and interference. CRISPR-Cas systems are currently classified into at least five different types, each with a signature protein among which Type III systems exhibit a dual DNA/RNA interference activity. Structures of a few Type III surveillance complexes have been determined: they are composed of several different subunits and exhibit striking architectural similarities to Type I surveillance complexes...
September 7, 2017: Current Issues in Molecular Biology
https://www.readbyqxmd.com/read/28878086/metsv-a-novel-archaeal-lytic-virus-targeting-methanosarcina-strains
#16
Katrin Weidenbach, Lisa Nickel, Horst Neve, Omer S Alkhnbashi, Sven Künzel, Anne Kupczok, Thorsten Bauersachs, Liam Cassidy, Andreas Tholey, Rolf Backofen, Ruth A Schmitz
A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as host. Due to its spherical morphology the virus was designated Methanosarcinaspherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs) all oriented in the same direction. Functions were predicted for some of these ORFs, i. e. like DNA polymerase, ATPase, DNA-binding protein, as well as envelope (structural) protein...
September 6, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28871175/fluorescent-crispr-adaptation-reporter-for-rapid-quantification-of-spacer-acquisition
#17
Lina Amlinger, Mirthe Hoekzema, E Gerhart H Wagner, Sanna Koskiniemi, Magnus Lundgren
CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I...
September 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28869593/how-type-ii-crispr-cas-establish-immunity-through-cas1-cas2-mediated-spacer-integration
#18
Yibei Xiao, Sherwin Ng, Ki Hyun Nam, Ailong Ke
CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes(1-5). Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer(6-9). Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical(7-9). The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle(6,10)...
September 4, 2017: Nature
https://www.readbyqxmd.com/read/28866660/generation-of-tryptophan-hydroxylase-2-gene-knockout-pigs-by-crispr-cas9-mediated-gene-targeting
#19
Ze Li, Hai-Yuan Yang, Ying Wang, Man-Ling Zhang, Xiao-Rui Liu, Qiang Xiong, Li-Ning Zhang, Yong Jin, Li-Sha Mou, Yan Liu, Rong-Feng Li, Yi Rao, Yi-Fan Dai
Unbalanced brain serotonin (5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2 (TPH2). In the present study, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at 38...
September 3, 2017: Journal of Biomedical Research
https://www.readbyqxmd.com/read/28865392/type-iii-crispr-cas-systems-when-dna-cleavage-just-isn-t-enough
#20
REVIEW
Nora C Pyenson, Luciano A Marraffini
Type III CRISPR-Cas systems have a unique targeting mechanism that requires the transcription of the DNA target and results in the degradation of not only the genome of the invader but also its transcripts. Here we discuss the most recent studies describing dual DNA and RNA targeting by these systems, as well as the implications of this complex molecular mechanism for immunity in vivo.
August 30, 2017: Current Opinion in Microbiology
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